DTPA prodrugs, compositions thereof, and methods of using the same

ABSTRACT

The present invention relates to trisodium diethylenetriamine pentaacetic acid (DTPA) prodrugs, such as, for example, DTPA di-ethyl esters. The invention further relates to compositions comprising DTPA prodrugs and methods of using the same.

RELATED APPLICATION DATA

This application is a continuation-in-part of International ApplicationNo. PCT/US2013/071738 filed on Nov. 25, 2013, which claims the benefitof and priority from U.S. Provisional Patent Application Ser. No.61/729,780, filed Nov. 26, 2012, the disclosures of each of which areincorporated herein by reference in their entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under Grant No.HHSN272201000030C awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to trisodium diethylenetriaminepentaacetic acid (DTPA) prodrugs. The present invention further relatesto compositions comprising DTPA prodrugs and methods of using the same.

BACKGROUND OF THE INVENTION

The presence of metals, such as radioactive and/or non-radioactivemetals, in an animal can be toxic to the body and/or cause negativehealth effects. Thus, removing such metals can be important to avoid orreduce toxicity to the animal.

Exposure to toxic metals can occur through environmental exposures. Forexample, an animal undergoing various medical procedures may be exposedto toxic metals. In addition, the United States and many other countriesface increasing threats from terrorist groups with respect to the use ofweapons of mass destruction against civilian populations. Of particularconcern is that some of these groups are intensifying their efforts toacquire and develop nuclear and radiological weapons, and there are alimited number of therapies that can be offered to victims of nuclearterrorism.

Currently, the only agents that have been approved by the U.S. Food andDrug Administration (FDA) as chelating agents for americium, curium andplutonium are the calcium and zinc salts of trisodium diethylenetriaminepentaacetic acid (DTPA). Transuranic radionuclides (i.e., those with anatomic number of 92 or greater), such as americium, curium andplutonium, can potentially be incorporated in radiation dispersaldevices (RDDs; “dirty bombs”). The primary goal in treating thoseexposed to transuranic radionuclides is to chelate the transuranicradionuclides before they become fixed in tissues, such as the liver andbone, and enhance their elimination from contaminated individuals.

SUMMARY OF THE INVENTION

A first aspect of the invention is a polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid characterized by a powder x-ray diffraction pattern substantiallythe same as that shown in FIG. 6A and/or a powder x-ray diffractionpattern having peaks at about 7.6, 12.4, 13.5, 14.0, 15.3, 18.1, 18.7,18.8, 21.0, 22.5, 23.4, 24.5, 28.7, and 35.7±0.2 degrees 2 theta.

A further aspect of the invention is a process of preparing a polymorphof6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid, comprising

(a) combining DTPA bis-anhydride, ethanol, and pyridine to form areaction mixture;

(b) stirring the reaction mixture under nitrogen for about 24 hours;

(c) adding the reaction mixture to dichloromethane to form adichloromethane solution;

(d) cooling the dichloromethane solution to a temperature of about −20°C. to form a precipitate of a polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid;

(e) filtering the dichloromethane solution to obtain the precipitate;

(f) optionally washing the precipitate with dichloromethane during thefiltering step; and

(g) optionally drying the precipitate, thereby obtaining the polymorph.

Another aspect of the invention is a polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid characterized by a powder x-ray diffraction pattern substantiallythe same as that shown in FIG. 6D and/or a powder x-ray diffractionpattern having peaks at about 8.1, 12.0, 13.8, 15.4, 16.0, 16.6, 18.3,19.3, 21.4, 22.1, 24.0, 26.5, and 29.2±0.2 degrees 2 theta.

Another aspect of the invention is a process of preparing a polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid, comprising

(a) combining DTPA bis-anhydride and absolute ethanol to form a reactionmixture;

(b) heating the reaction mixture to reflux while stirring for about 1.5hours;

(c) filtering the reaction mixture to form a filtrate;

(d) cooling the filtrate to a temperature below about 20° C. to form aprecipitate of a polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid;

(e) filtering the filtrate to obtain the precipitate;

(f) optionally washing the precipitate with cold ethanol;

(g) optionally washing the precipitate with methyl tert-butyl ether toform a filter cake;

(h) optionally mixing the filter cake with ethanol to form a secondslurry;

(i) optionally heating the second slurry to a temperature of about 70°C.;

(j) optionally filtering the second slurry to form a second filtrate;

(k) optionally cooling the second filtrate to a temperature below about20° C. to form a second precipitate;

(l) optionally filtering the second filtrate to obtain the secondprecipitate; and

(m) optionally drying the precipitate, thereby obtaining the polymorph.

A further aspect of the invention is a method of treating a subject toremove a radioactive element and/or a non-radioactive element from thesubject comprising: administering a therapeutically effective amount ofa DTPA prodrug of the present invention to a subject. In someembodiments, the DTPA prodrug is a DTPA di-ethyl ester of the presentinvention, such as, but not limited to, a DTPA di-ethyl ester polymorphof the present invention.

A further aspect of the invention is a method of increasing the amountof a radioactive element and/or a non-radioactive element removed from asubject comprising: administering a therapeutically effective amount ofa DTPA prodrug of the present invention to a subject. In someembodiments, the DTPA prodrug is a DTPA di-ethyl ester of the presentinvention, such as, but not limited to, a DTPA di-ethyl ester polymorphof the present invention.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a synthetic scheme for C2E2.

FIG. 2 shows samples of dosing solutions containing C2E2 from Lot 050,from left to right: 20 mg/mL, 60 mg/mL and 100 mg/mL C2E2 dosingsolutions.

FIG. 3 shows differential scanning calorimetry (DSC) data for C2E2preparations.

FIG. 4 shows the comparison of the thermogravametric data obtained forboth C2E2 batches. Method 1 is the dotted line and Method 2 is the solidline.

FIG. 5 shows a graph of the concentrations of metal ions in C2E2preparations as detected by inductively coupled plasma mass spectrometry(ICP-MS).

FIGS. 6A-6E show X-ray diffraction (XRD) patterns for C2E2 preparations:A) Reference standard; B) MTD Lot; C) Lot 005-2; D) Lot 050; and E) acomparison of XRD patterns A-D.

FIG. 7 shows an overlay of the XRD patterns for A) reference standardand B) MTD Lot.

FIG. 8 shows an overlay of the XRD patterns for C) Lot 005-2 and D) Lot050.

FIG. 9 shows the scanning electron micrographs of C2E2 preparations.Left: C2E2 MTD Lot; right: C2E2 Lot 050.

FIG. 10 shows the chromatogram of C2E2 of Lot 050.

FIG. 11 shows the chromatogram of C2E2 crystals produced from water.

FIG. 12 shows the chromatogram of C2E2 solid material from 1 hourheating in ethanol.

FIG. 13 shows the chromatogram of C2E2 filtrate from 1 hour heating inethanol.

FIG. 14 shows the cumulative transport of C2E2 from the apical tobasolateral compartment over two hours. Error bars represent the 95%confidence interval.

FIG. 15 shows the change in flux at increasing concentrations of C2E2.Error bars represent the 95% confidence interval.

FIG. 16 shows the change in apparent permeation coefficient withincreasing concentration of C2E2. Error bars represent the 95%confidence interval.

FIG. 17 shows the daily ²⁴¹Am clearance in male Sprague Dawley rats.

FIG. 18 shows the daily ²⁴¹Am clearance in female Sprague Dawley rats.

FIG. 19 shows the total decorporation C2E2 dose response curve showingtotal ²⁴¹Am decorporation in male and female Sprague Dawley rats sevendays after contamination.

FIG. 20 shows the liver burden C2E2 dose response curve showing ²⁴¹Amliver burden in male and female Sprague Dawley rats seven days aftercontamination.

FIG. 21 shows the skeletal burden C2E2 dose response curve showing ²⁴¹Amliver burden in male and female Sprague Dawley rats seven days aftercontamination.

FIG. 22 shows the wound site burden C2E2 dose response curve showing²⁴¹Am content at the wound site in male and female Sprague Dawley rats.

FIG. 23A shows the average percent of recovered activity of ²⁴¹Am inurine per day in dogs after administration of ²⁴¹Am by inhalationexposure.

FIG. 23B shows the average percent of recovered activity of ²⁴¹Am infeces per day in dogs after administration of ²⁴¹Am by inhalationexposure.

FIG. 24A shows the average percentage of recovered activity of ²⁴¹Am forcumulative urinary excretion of dogs administered different doses ofC2E2 at 24 h after ²⁴¹Am inhalation exposure.

FIG. 24B shows the average percentage of recovered activity of ²⁴¹Am forcumulative fecal excretion of dogs administered different doses of C2E2at 24 h after ²⁴¹Am inhalation exposure.

FIGS. 25A-D show the average percentage of recovered activity of ²⁴¹Amin A) liver, B) spleen, C) kidney, and D) lung of dogs administereddifferent doses of C2E2 at 24 h after ²⁴¹Am inhalation exposure.

FIGS. 26A-F show the average percentage of recovered activity of ²⁴¹Amin A) ovaries, B) testes, C) GIT, D) TBLN, E) soft tissue, and F) totalbone content in dogs administered different doses of C2E2 at 24 h after²⁴¹Am inhalation exposure.

FIG. 27 shows the relative concentration of C2E2 over time in samplesprepared at 60, 70 and 100 mg/mL.

FIGS. 28A-C show the stability of C2E2 in A) rat, B) dog, and C) humanplasma with each column in the graph representing (from left to right)normal plasma; heat inactivated plasma; a control substrate, diltiazem,in normal plasma; and the control substrate, diltiazem, in heatinactivated plasma over time.

FIG. 29 shows the americium burden in liver, skeletal, and injectionsite tissue seven days after contamination with each column in the graphrepresenting (from left to right) control, C2E2 once daily (OD), C2E2twice daily (BD), and DTPA.

FIGS. 30A and 30B show the percentage of initial americium injectioneliminated in the urine per hour with A) showing the profile over theduration of the whole study and B) showing the expanded view starting 24hours after americium contamination as the first C2E2 doses wereadministered. The percent decorporation per hour at time zero reflectsthe average hourly decorporation over the first 24 hours of the study. ♦is the untreated control group, ▪ is the C2E2 600 mg/kg OD group, ▴ isthe C2E2 300 mg/kg BD group.

FIG. 31 shows a titration curve for C2E2 with 1 N potassium hydroxide.

FIG. 32 shows a titration curve for C2E2 (solid line) and C2E2-Gd(dotted line) with 0.1 N hydrochloric acid.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter. Thisinvention may be embodied in different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting of the invention. As used in the description and the appendedclaims, the singular forms “a”, “an” and “the” are intended to includethe plural forms as well, unless the context clearly indicatesotherwise.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. The terminology used inthe description herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

All patents, patent applications and publications referred to herein areincorporated by reference in their entirety for the teachings relevantto the sentence and/or paragraph in which the reference is presented. Incase of a conflict in terminology, the present specification iscontrolling.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the embodiments of the invention describedherein may be used in any combination. For example, features describedin relation to one embodiment may also be applicable to and combinablewith other embodiments and aspects of the invention.

Moreover, the embodiments of the present invention also contemplate thatin some embodiments, any feature or combination of features set forthherein may be excluded or omitted. To illustrate, if the specificationstates that a complex comprises components A, B and C, in someembodiments, any of A, B or C, or a combination thereof, may be omittedand disclaimed.

As used herein, the transitional phrase “consisting essentially of” (andgrammatical variants) is to be interpreted as encompassing the recitedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention. See, In re Herz,537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in theoriginal); see also MPEP §2111.03. Thus, the term “consistingessentially of” as used herein should not be interpreted as equivalentto “comprising.”

The term “about,” as used herein when referring to a measurable value,such as, for example, an amount or concentration, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount. A range provided herein for a measureable value mayinclude any other range and/or individual value therein.

“Substituted” as used herein to describe chemical structures, groups, ormoieties, refers to the structure, group, or moiety comprising one ormore substituents. As used herein, in cases in which a first group is“substituted with” a second group, the second group is attached to thefirst group whereby a moiety of the first group (typically a hydrogen)is replaced by the second group. The substituted group may contain oneor more substituents that may be the same or different.

“Substituent” as used herein references a group that replaces anothergroup in a chemical structure. Typical substituents include nonhydrogenatoms (e.g., halogens), functional groups (such as, but not limited to,amino, sulfhydryl, carbonyl, hydroxyl, alkoxy, carboxyl, silyl,silyloxy, phosphate and the like), hydrocarbyl groups, and hydrocarbylgroups substituted with one or more heteroatoms. Exemplary substituentsinclude, but are not limited to, alkyl, lower alkyl, halo, haloalkyl,alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo,heterocycloalkyl, aryl, arylalkyl, lower alkoxy, thioalkyl, hydroxyl,thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silylalkyl,silyloxy, boronyl, and modified lower alkyl.

“Alkyl” as used herein alone or as part of another group, refers to astraight or branched chain hydrocarbon containing from 1 to 30 carbonatoms. In some embodiments, the alkyl group may contain 1, 2, or 3 up to4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. Representative examplesof alkyl include, but are not limited to, methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl,isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.“Lower alkyl” as used herein, is a subset of alkyl and refers to astraight or branched chain hydrocarbon group containing from 1 to 4carbon atoms. Representative examples of lower alkyl include, but arenot limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,tert-butyl, and the like. The term “alkyl” or “loweralkyl” is intendedto include both substituted and unsubstituted alkyl or loweralkyl unlessotherwise indicated and these groups may be substituted with groups suchas, but not limited to, polyalkylene oxides (such as PEG), halo (e.g.,haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl,hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethyleneglycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy,cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy,heterocyclolalkyloxy, mercapto, alkyl-S(O)_(m), haloalkyl-S(O)_(m),alkenyl-S(O)_(m), alkynyl-S(O)_(m), cycloalkyl-S(O)_(m),cycloalkylalkyl-S(O)_(m), aryl-S(O)_(m), arylalkyl-S(O)_(m),heterocyclo-S(O)_(m), heterocycloalkyl-S(O)_(m), amino, carboxy,alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino,cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino,heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester,amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano,where m=0, 1, 2 or 3.

“Alkenyl” as used herein alone or as part of another group, refers to astraight or branched chain hydrocarbon containing from 1 to 30 carbonatoms (or in loweralkenyl 1 to 4 carbon atoms) which include 1 to 10double bonds in the hydrocarbon chain. In some embodiments, the alkenylgroup may contain 1, 2, or 3 up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbonatoms. Representative examples of alkenyl include, but are not limitedto, methylene (═CH₂), vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), 2-butenyl,3-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene,and the like. The term “alkenyl” or “loweralkenyl” is intended toinclude both substituted and unsubstituted alkenyl or loweralkenylunless otherwise indicated and these groups may be substituted withgroups such as those described in connection with alkyl and loweralkylabove.

“Alkynyl” as used herein alone or as part of another group, refers to astraight or branched chain hydrocarbon containing from 1 to 30 carbonatoms (or in loweralkynyl 1 to 4 carbon atoms) which include at leastone triple bond in the hydrocarbon chain. In some embodiments, thealkynyl group may contain 2, or 3 up to 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 carbon atoms. Representative examples of alkynyl include, but are notlimited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl,and the like. The term “alkynyl” or “loweralkynyl” is intended toinclude both substituted and unsubstituted alkynyl or loweralkynylunless otherwise indicated and these groups may be substituted with thesame groups as set forth in connection with alkyl and loweralkyl above.

“Cycloalkyl” as used herein alone or as part of another group, refers toa saturated or partially unsaturated cyclic hydrocarbon group containingfrom 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in aheterocyclic group as discussed below). Representative examples ofcycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. These rings may be optionally substitutedwith additional substituents as described herein such as halo orloweralkyl. The term “cycloalkyl” is generic and intended to includeheterocyclic groups as discussed below unless specified otherwise.

“Aryl” as used herein alone or as part of another group, refers to amonocyclic carbocyclic ring system or a bicyclic carbocyclic fused ringsystem or higher having one or more aromatic rings. Representativeexamples of aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl,tetrahydronaphthyl, and the like. The term “aryl” is intended to includeboth substituted and unsubstituted aryl unless otherwise indicated andthese groups may be substituted with the same groups as set forth inconnection with alkyl and loweralkyl above.

“Arylalkyl” as used herein alone or as part of another group, refers toan aryl group, as defined herein, appended to the parent molecularmoiety through an alkyl group, as defined herein. Representativeexamples of arylalkyl include, but are not limited to, benzyl,2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

“Amino acid derivative” as used herein, refers to an amino acidsubstituted with one or more substituents. Exemplary substituentsinclude, but are not limited to, alkyl, lower alkyl, halo, haloalkyl,alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo,heterocycloalkyl, aryl, arylalkyl, lower alkoxy, thioalkyl, hydroxyl,thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silylalkyl,silyloxy, boronyl, and modified lower alkyl. Exemplary amino acidderivatives include, but are not limited to, alanine methyl ester,alanine ethyl ester, alanine tert-butyl ester, valine methyl ester,valine ethyl ester, valine tert-butyl ester, phenylalanine methyl esterphenylalanine ethyl ester, phenylalanine tert-butyl ester,phenylalainamide, N-acetyl-phenylalanine,N-ethoxycarbonyl-phenylalanine, tyrosine methyl ester, tyrosine ethylester, tyrosine tert-butyl ester, N-acetyl-tyrosine, andO-benzyl-tyrosine.

Described herein are DTPA prodrugs of Formula (I)

wherein:

R is —OR¹ or —NHR¹; and

R¹ is each independently selected from the group consisting of H, C₁-C₃₀alkyl, C₂-C₃₀ alkenyl, C₂-C₃₀ alkynyl, benzyl, cycloalkyl, aryl,arylalkyl, arylalkenyl, arylalkynyl, heterocyclic, and amino acidderivative and wherein when R is —OR¹ at least one R¹ is not hydrogen.

In particular embodiments of the present invention, R is —OR¹ and R¹ isC₁-C₃₀ alkyl, e.g., C₁-C₁₂ and/or C₁-C₆. In certain embodiments of thepresent invention, the DTPA prodrug of Formula (I) is penta-substituted.When one or more R¹ is present, then R¹ at each occurrence may be thesame as another R¹ and/or different than another R¹. Thus, all R¹ may bethe same, all R¹ may be different, or some R¹ may be the same and someR¹ may be different. A DTPA prodrug of Formula (I) (i.e., a DTPA prodrugof the present invention) does not include trisodium diethylenetriaminepentaacetic acid (DTPA). Exemplary DTPA prodrugs of Formula (I) andtheir synthesis can be found in U.S. Pat. No. 8,030,358, which isincorporated herein by reference in its entirety. In some embodiments ofthe present invention, a DTPA prodrug of the present invention is a DTPAdi-ethyl ester. In some embodiments of the present invention, a DTPAprodrug of the present invention has one of the following structures:

In particular embodiments of the present invention, a DTPA prodrug ofFormula (I) has certain physical-chemical properties. For example, theDTPA prodrug of Formula (I) may have a molecular weight from about 400to about 700 or any range and/or individual value therein, such as fromabout 400 to about 600 or from about 400 to about 500. In someembodiments of the present invention, the DTPA prodrug of Formula (I)may have an apparent solubility of about 100 mg/mL to about 200 mg/mL inan aqueous solution at a pH in a range of about 2 to about 3. In someembodiments of the present invention, the DTPA prodrug of Formula (I)may have an apparent solubility of about 150 mg/mL in an aqueoussolution at a pH in a range of about 2 to about 3. Thus, the DTPAprodrug of Formula (I) may have a log P value of about −3.5 to about−1.5 at a pH of about 3.0, and in some embodiments a log P value ofabout −2.9 to about −2.1 at a pH of about 3.0.

In some embodiments, a DTPA prodrug of the present invention has abioavailability of greater than 5%. In some embodiments, a DTPA prodrugof the present invention has a bioavailability of greater than 5% uponadministration to a subject, such as, for example, when orallyadministered to a subject. In some embodiments of the present invention,a DTPA prodrug may be administered to a subject and may be resistant tohydrolysis prior to absorption in the gastrointestinal (GI) tract of thesubject. In some embodiments of the present invention, the DTPA prodrugmay have an apparent solubility of about 100 mg/mL to about 200 mg/mL inan aqueous solution at a pH in a range of about 2 to about 3 and maydissolve rapidly during transit through the GI tract of a subject. Insome embodiments of the present invention, the dissolution of the DTPAprodrug in the GI tract of a subject may allow for absorption of theDTPA prodrug to occur.

In some embodiments, the present invention provides a polymorph of aDTPA di-ethyl ester having the chemical name6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid (also referred to herein as C2E2) and having the followingstructure:

In some embodiments of the present invention, a polymorph of a DTPAdi-ethyl ester of the present invention may be more stable than otherknown forms of C2E2. Stability can be measured by methods known to thoseof skill in the art. For example, in some embodiments, stability isdetermined by comparing the melting points of C2E2 polymorphs. In someembodiments of the present invention, a C2E2 polymorph of the presentinvention is more stable compared to other C2E2 polymorphs since theC2E2 polymorph of the present invention has a higher melting point thanthe melting points of the other C2E2 polymorphs. Stability may refer tothe stability of a polymorph of the present invention during storageand/or in a solution, such as an aqueous solution.

In certain embodiments of the present invention, a polymorph of a DTPAdi-ethyl ester of the present invention may be easier to formulateand/or prepare compared to other known forms of C2E2. For example, apolymorph of a DTPA di-ethyl ester of the present invention may have animproved solubility compared to other known forms of C2E2. In someembodiments of the present invention, a polymorph of a DTPA di-ethylester of the present invention may provide a better bioavailabilityafter delivery to a subject compared to other known forms of C2E2 whenadministered in the same manner.

In certain embodiments of the present invention, a DTPA prodrug of thepresent invention may be de-esterified to form DTPA and/or a metaboliteof C2E2, such as, but not limited to, a DTPA mono-ethyl ester (e.g.,C2E1). In some embodiments of the present invention, a DTPA di-ethylester of the present invention may be a DTPA prodrug that may bede-esterified by an esterase to form DTPA and/or a metabolite of C2E2.Alternatively or in addition, in certain embodiments of the presentinvention, a DTPA di-ethyl ester of the present invention may be achelating agent, such as, but not limited to, a chelating agent forradioactive elements and/or non-radioactive elements.

According to some embodiments of the present invention, a polymorph of aDTPA di-ethyl ester of the present invention may have a powder x-raydiffraction pattern corresponding to (e.g., substantially the same as,the same as, etc.) a powder x-ray diffraction pattern shown in any ofFIGS. 6-8. In particular embodiments of the present invention, apolymorph of a DTPA di-ethyl ester of the present invention has a powderx-ray diffraction pattern corresponding to the powder x-ray diffractionpattern in FIGS. 6 A, B, C, and/or D.

In certain embodiments of the present invention, a polymorph of a DTPAdi-ethyl ester of the present invention has a powder x-ray diffractionpattern corresponding to the powder x-ray diffraction pattern in FIG.6A. The polymorph having a powder x-ray diffraction patterncorresponding to FIG. 6A is referred to herein as Form I. A DTPAdi-ethyl ester of Form I may be characterized by a powder x-raydiffraction pattern having peaks at about 7.6, 12.4, 13.5, 14.0, 15.3,18.1, 18.7, 18.8, 21.0, 22.5, 23.4, 24.5, 28.7, and 35.7±0.2 degrees 2theta.

A DTPA di-ethyl ester of Form I is characterized by a melting point fromabout 109° C. to about 122° C., or any range and/or individual valuetherein, as measured using differential scanning calorimetry over arange of about 25° C. to about 320° C. with a heating rate of about10.00° C./min. The rate, for example, may be between 8.00° C./min and12.00° C./min or any other range and/or individual value therein. Insome embodiments of the present invention, a DTPA di-ethyl ester of FormI may have a melting point of about 109° C., 110° C., 111° C., 112° C.,113° C., 114° C., 115° C., 116° C., 117° C., 118° C., 119° C., 120° C.,121° C., or 122° C., or any range therein. In certain embodiments of thepresent invention, a DTPA di-ethyl ester of Form I may have a meltingpoint at about 111.8° C., 116.3° C., or 119.4° C.

DTPA di-ethyl ester of Form I may be obtained by precipitating DTPAdi-ethyl ester in dichloromethane. In some embodiments, a DTPA di-ethylester of Form I is obtained by precipitating DTPA di-ethyl ester indichloromethane while cooling at a temperature of about −20° C. Thetemperature, for example, may be between −16° C. and −24° C. or anyother range and/or individual value therein.

According to some embodiments of the present invention, provided is amethod of preparing a DTPA di-ethyl ester of Form I, the methodcomprising combining DTPA bis-anhydride, ethanol and pyridine to form areaction mixture, stirring the reaction mixture under nitrogen or otherinert gas(es) for, for example, about 24 hours, such as, for example,between 20 hours and 28 hours or any other range and/or individual valuetherein, adding the reaction mixture to dichloromethane to form adichloromethane solution, cooling the dichloromethane solution to atemperature of about −20° C., such as, for example, between −16° C. and−24° C. or any other range and/or individual value therein, to form aprecipitate of a polymorph of a DTPA di-ethyl ester of the presentinvention, filtering the dichloromethane solution to obtain theprecipitate, optionally washing the precipitate with dichloromethaneduring the filtering step, and optionally drying the precipitate,thereby obtaining a DTPA di-ethyl ester of Form I.

In some embodiments of the present invention, a DTPA di-ethyl ester ofForm I may be obtained during and/or after the cooling step in a methodof preparing a DTPA di-ethyl ester of Form I of the present invention.In certain embodiments of the present invention, a method of preparing aDTPA di-ethyl ester of Form I of the present invention comprises washingthe precipitate with dichloromethane during the filtering step anddrying the precipitate, and a DTPA di-ethyl ester of Form I may beobtained during and/or after the drying step.

In other embodiments of the present invention, a polymorph of a DTPAdi-ethyl ester of the present invention has a powder x-ray diffractionpattern corresponding to the powder x-ray diffraction pattern in FIG.6D. The polymorph having a powder x-ray diffraction patterncorresponding to FIG. 6D is referred to herein as Form II. A DTPAdi-ethyl ester of Form II may be characterized by a powder x-raydiffraction pattern having peaks at about 8.1, 12.0, 13.8, 15.4, 16.0,16.6, 18.3, 19.3, 21.4, 22.1, 24.0, 26.5, and 29.2±0.2 degrees 2 theta.

A DTPA di-ethyl ester of Form II is characterized by a melting pointfrom about 132° C. to about 143° C., or any range and/or individualvalue therein, as measured using differential scanning calorimetry overa range of about 25° C. to about 320° C. with a heating rate of about10.00° C./min. The rate, for example, may be between 8.00° C./min and12.00° C./min or any other range and/or individual value therein. Insome embodiments of the present invention, a DTPA di-ethyl ester of FormII may have a melting point of about 132° C., 133° C., 134° C., 135° C.,136° C., 137° C., 138° C., 139° C., 140° C., 141° C., 142° C., or 143°C., or any range therein. In certain embodiments of the presentinvention, a DTPA di-ethyl ester of Form II may have a melting point atabout 134.8° C., 136.4° C., 139.1° C., or 141.6° C.

DTPA di-ethyl ester of Form II may be obtained by precipitating DTPAdi-ethyl ester in ethanol. In some embodiments, a DTPA di-ethyl ester ofForm II is obtained by precipitating DTPA di-ethyl ester in ethanolwhile cooling to a temperature below about 20° C. The temperature, forexample, may be between 16° C. and 24° C. or any other range and/orindividual value therein.

According to some embodiments of the present invention, provided is amethod of preparing a DTPA di-ethyl ester of Form II, the methodcomprising combining DTPA bis-anhydride and absolute ethanol to form areaction mixture, heating the reaction mixture to reflux while stirringfor, for example, about 1.5 hours, such as, for example, between 1.2hours and 1.8 hours or any other range and/or individual value therein,filtering the reaction mixture to form a filtrate, cooling the filtrateto a temperature below about 20° C., such as, for example, between 16°C. and 24° C. or any other range and/or individual value therein, toform a precipitate of a polymorph of a DTPA di-ethyl ester of thepresent invention, filtering the filtrate to obtain the precipitate, andoptionally drying the precipitate, thereby obtaining DTPA di-ethyl esterof Form II. In certain embodiments, following the filtering step andprior to the optional drying step, the method may further comprise oneor more steps of washing the precipitate with cold ethanol and thenmethyl tert-butyl ether (MTBE) to form a filter cake, mixing the filtercake with ethanol to form a second slurry, heating the second slurry toa temperature of about 70° C., such as, for example, between 56° C. and84° C. or any other range and/or individual value therein, filtering thesecond slurry to form a second filtrate, cooling the second filtrate toa temperature below about 20° C., such as, for example, between 16° C.and 24° C. or any other range and/or individual value therein, to form asecond precipitate of a polymorph of a DTPA di-ethyl ester of thepresent invention, filtering the second filtrate to obtain theprecipitate, and then optionally drying the precipitate, therebyobtaining DTPA di-ethyl ester of Form II.

In some embodiments of the present invention, a DTPA di-ethyl ester ofForm II may be obtained during and/or after the first cooling step in amethod of preparing a DTPA di-ethyl ester of Form II of the presentinvention. In certain embodiments of the present invention, a DTPAdi-ethyl ester of Form II may be obtained during and/or after the secondcooling step in a method of preparing a DTPA di-ethyl ester of Form IIof the present invention. In some embodiments of the present invention,a method of preparing a DTPA di-ethyl ester of Form II of the presentinvention comprises drying the first and/or second precipitate, and aDTPA di-ethyl ester of Form II may be obtained during and/or after thedrying step.

According to another aspect of the present invention, provided hereinare pharmaceutical compositions comprising a DTPA prodrug of the presentinvention. In some embodiments, the DTPA prodrug is a DTPA di-ethylester of the present invention, such as, but not limited to, a polymorphof a DTPA di-ethyl ester of the present invention. In certainembodiments, a pharmaceutical composition of the present inventioncomprises a DTPA di-ethyl ester of Form I and/or Form II. One or moredifferent DTPA prodrugs of the present invention may be present in apharmaceutical composition of the present invention. The pharmaceuticalcomposition may comprise at least about 50% or more of one DTPA prodrug(e.g., a DTPA di-ethyl ester of Form I or Form II) compared to the totalamount of the one or more different DTPA prodrugs present in thecomposition, and, in some embodiments, at least about 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more of one DTPA prodrugcompared to the total amount of the one or more different DTPA prodrugspresent in the composition. In some embodiments, a pharmaceuticalcomposition of the present invention further comprises apharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier is any carrier which is relatively non-toxic and innocuous to asubject at concentrations consistent with effective activity of a DTPAprodrug of the present invention so that any side effects ascribable tothe carrier do not vitiate the beneficial effects of the DTPA prodrug.

A pharmaceutical composition of the present invention may be formulatedto administer in a single dose and/or unit about 1 mg to about 2,000 mgof a DTPA prodrug of the present invention per kilogram of a subject'stotal body weight, or any range and/or individual value therein, suchas, but not limited to about 10 mg to about 1,000 mg, about 1,000 mg toabout 2,000 mg, about 200 mg to about 600 mg, about 1 mg to about 500mg, about 5 mg to about 250 mg, about 5 mg to about 100 mg, about 15 mgto about 45 mg, or about 10 mg to about 40 mg per kilogram of asubject's total body weight. A DTPA prodrug of the present invention maybe administered with a pharmaceutically acceptable carrier using anyeffective conventional dosage unit form, such as, but not limited to,immediate and timed release preparations, orally, parenterally,topically, or the like. Exemplary pharmaceutical compositions include,but are not limited to, those described in U.S. Pat. No. 8,030,358,International Application No. PCT/US12/60985, and U.S. PatentApplication Publication No. 2014/0243411, the contents of each of whichare incorporated herein by reference in their entirety for the contentsrelated to formulations and drug delivery systems and methods ofpreparing such formulations and systems. In certain embodiments of thepresent invention, a pharmaceutical composition of the present inventionis suitable for oral administration.

For oral administration, a DTPA prodrug of the present invention may beformulated into solid or liquid preparations such as, but not limitedto, capsules, pills, tablets, troches, lozenges, chewing gum, melts,powders, solutions, suspensions, or emulsions, and may be preparedaccording to methods known to the art for the manufacture ofpharmaceutical compositions. The solid unit dosage forms may be acapsule which can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricants, and inert fillers suchas lactose, sucrose, calcium phosphate, and corn starch.

In another embodiment of the present invention, a DTPA prodrug of thepresent invention may be tableted with conventional tablet bases such aslactose, sucrose, and cornstarch in combination with binders such asacacia, cornstarch, or gelatin; disintegrating agents intended to assistthe break-up and dissolution of the tablet following administration suchas potato starch, alginic acid, corn starch, and guar gum; lubricantsintended to improve the flow of tablet granulation and to prevent theadhesion of tablet material to the surfaces of the tablet dies andpunches, for example, talc, stearic acid, or magnesium, calcium or zincstearate; dyes; coloring agents; and flavoring agents intended toenhance the aesthetic qualities of the tablets and make them moreacceptable to the patient. Suitable excipients for use in oral liquiddosage forms include diluents such as water and alcohols, for example,ethanol, benzyl alcohol, and polyethylene alcohols, either with orwithout the addition of a pharmaceutically acceptable surfactant,suspending agent, or emulsifying agent. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance tablets, pills or capsules may be coated withshellac, sugar or both.

Dispersible powders and granules are suitable for the preparation of anaqueous suspension. They may provide a DTPA prodrug of the presentinvention in admixture with a dispersing or wetting agent, a suspendingagent, and/or one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those already mentionedabove. Additional excipients, for example, those sweetening, flavoringand coloring agents described above, may also be present.

A pharmaceutical composition of the present invention may also be in theform of oil-in-water emulsions. The oily phase may be a vegetable oilsuch as liquid paraffin or a mixture of vegetable oils. Suitableemulsifying agents may be (1) naturally occurring gums such as gumacacia and gum tragacanth, (2) naturally occurring phosphatides such assoy bean and lecithin, (3) esters or partial esters derived from fattyacids and hexitol anhydrides, for example, sorbitan monooleate, and (4)condensation products of said partial esters with ethylene oxide, forexample, polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening and flavoring agents.

Oily suspensions may be formulated by suspending a DTPA prodrug of thepresent invention in a vegetable oil such as, for example, arachis oil,olive oil, sesame oil, or coconut oil; or in a mineral oil such asliquid paraffin. The oily suspensions may contain a thickening agentsuch as, for example, beeswax, hard paraffin, or cetyl alcohol. Thesuspensions may also contain one or more preservatives, for example,ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents; one ormore flavoring agents; and one or more sweetening agents such as sucroseor saccharin.

Syrups and elixirs may be formulated with sweetening agents such as, forexample, glycerol, propylene glycol, sorbitol, or sucrose. Suchformulations may also contain a demulcent, and preservative, flavoringand coloring agents.

A DTPA prodrug of the present invention may also be administeredparenterally, that is, subcutaneously, intravenously, intramuscularly,or interperitoneally, as injectable dosages of the DTPA prodrug in aphysiologically acceptable diluent with a pharmaceutical carrier whichmay be a sterile liquid or mixture of liquids such as water, saline,aqueous dextrose and related sugar solutions; an alcohol such asethanol, isopropanol, or hexadecyl alcohol; glycols such as propyleneglycol or polyethylene glycol; glycerol ketals such as2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such aspoly(ethyleneglycol) 400; an oil; a fatty acid; a fatty acid ester orglyceride; or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant such as a soap or adetergent, suspending agent such as pectin, carbomers, methycellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagent and other pharmaceutical adjuvants.

Illustrative of oils which may be used in the parenteral formulations ofthis invention are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, sesame oil, cottonseedoil, corn oil, olive oil, petrolatum, and mineral oil. Suitable fattyacids include oleic acid, stearic acid, and isostearic acid. Suitablefatty acid esters are, for example, ethyl oleate and isopropylmyristate. Suitable soaps include fatty alkali metal, ammonium, andtriethanolamine salts and suitable detergents include cationicdetergents, for example, dimethyl dialkyl ammonium halides, alkylpyridinium halides, and alkylamine acetates; anionic detergents, forexample, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, andmonoglyceride sulfates, and sulfosuccinates; nonionic detergents, forexample, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylenepolypropylene copolymers; and amphoteric detergents, forexample, alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternaryammonium salts, as well as mixtures.

A parenteral composition of the present invention may contain from about0.5% to about 90% or more by weight of a DTPA prodrug of the presentinvention in solution. Preservatives and buffers may also be usedadvantageously. In order to minimize or eliminate irritation at the siteof injection, such compositions may contain a non-ionic surfactanthaving a hydrophile-lipophile balance (HLB) of from about 12 to about17. The quantity of surfactant in such formulation ranges from about 5%to about 15% by weight. The surfactant can be a single component havingthe above HLB or can be a mixture of two or more components having thedesired HLB.

Illustrative of surfactants used in parenteral formulations are theclass of polyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

A pharmaceutical composition of the present invention may be in the formof sterile injectable aqueous suspensions. Such suspensions may beformulated according to known methods using suitable dispersing orwetting agents and suspending agents such as, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents which may be a naturally occurringphosphatide such as lecithin, a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate, acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethyleneoxycetanol, a condensationproduct of ethylene oxide with a partial ester derived form a fatty acidand a hexitol such as polyoxyethylene sorbitol monooleate, or acondensation product of an ethylene oxide with a partial ester derivedfrom a fatty acid and a hexitol anhydride, for example polyoxyethylenesorbitan monooleate.

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent. Diluents and solvents that may be employed are, for example,water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils are conventionally employed as solvents orsuspending media. For this purpose, any bland, fixed oil may be employedincluding synthetic mono or diglycerides. In addition, fatty acids suchas oleic acid may be used in the preparation of injectables.

A further aspect of the present invention provides a method of removinga radioactive element and/or non-radioactive element from a subjectcomprising administering a DTPA prodrug and/or composition of thepresent invention. “Radioactive element” as used herein, refers to achemical element that emits particulate radiation such as, but notlimited to, alpha particles, beta particles, Auger electrons, etc., or achemical element that emits photons such as, but not limited to, x-rays,gamma rays, etc. The radioactive element may be present in its elementalform or as part of a chemical compound. The radioactive element can havean atomic number of 1 to 103. In certain embodiments of the presentinvention, the radioactive element is in the actinide series (i.e., hasan atomic number of 89-103) of elements. In particular embodiments, theradioactive element is an isotope of plutonium (Pu), americium (Am), orcurium (Cm). “Non-radioactive element” as used herein refers to achemical element that is not a radioactive element and has an atomicnumber of 1 to 103. In certain embodiments, the non-radioactive elementis a heavy metal and/or an element present in a subject at a dosedetrimental and/or toxic to the subject. Exemplary non-radioactiveelements include, but are not limited to, lead, mercury, plutonium,vanadium, tungsten, cadmium, arsenic, zinc, copper, manganese, selenium,chromium, molybdenum, aluminum, bismuth, gold, gallium, gadolinium,lithium, silver, cobalt, iron, nickel, selenium, thallium, and anycombination thereof.

In some embodiments, the radioactive element and/or non-radioactiveelement is in ionic form and/or is bound and/or complexed to a chemicalmoiety. For example, in some embodiments, gadolinium ions, lineargadolinium-based contrast agents (GBCAs), and/or macrocyclic GBCAs maybe removed from a subject by administering a DTPA prodrug of the presentinvention, such as, but not limited to, C2E2. A DTPA prodrug of thepresent invention and/or DTPA may bind and/or be complexed to aradioactive and/or non-radioactive element to facilitate its removalfrom the subject. In some embodiments, a DTPA prodrug of the presentinvention and/or DTPA may bind and/or be complexed to a radioactiveand/or non-radioactive element in the bloodstream of a subject and/or inone or more tissues of a subject. In some embodiments, a DTPA prodrug ofthe present invention and/or DTPA may remove a radioactive and/ornon-radioactive element through urinary and/or fecal excretion.

The term “administering”, “administration”, and grammatical variantsthereof, as used herein, refer to any mode of delivery to a subject. ADTPA prodrug of the present invention may be administered to a subjectby any suitable route, including, but not limited to, orally (inclusiveof administration via the oral cavity), parenterally, by inhalationspray, topically, transdermally, rectally, nasally, sublingually,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. In certain embodiments of the present invention, a DTPAprodrug and/or composition of the present invention is administeredorally.

Another aspect of the present invention provides a method of treating asubject comprising administering a DTPA prodrug and/or composition ofthe present invention to the subject. The term “treating” andgrammatical variants thereof, as used herein, refer to any type oftreatment that imparts a benefit to a subject, including delaying and/orreducing the onset and/or progression of one or more symptom(s) and/orcondition(s), reducing the severity of one or more symptom(s) and/orcondition(s), etc. Those skilled in the art will appreciate that thebenefit imparted by the treatment according to the methods of thepresent invention is not necessarily meant to imply cure (e.g., nodetectable incorporation of a radioactive and/or non-radioactive elementinto a subject's tissue, such as, for example, bones, and/or organs)and/or abolition of the symptom(s) and/or condition(s). The symptomsand/or conditions may include those occurring after exposure of thesubject to a radioactive and/or non-radioactive element.

In some embodiments of the present invention, a method of treating asubject exposed to a radioactive and/or non-radioactive element isprovided comprising administering a DTPA prodrug and/or composition ofthe present invention. Thus, in some embodiments of the presentinvention, methods are provided for the removal of a radioactive and/ornon-radioactive element from a subject exposed to the radioactiveelement and/or non-radioactive element. Those skilled in the art willappreciate that the removal of the radioactive and/or non-radioactiveelement from the subject may be partial or complete.

Another aspect of the present invention provides a method of preventingthe incorporation of a radioactive and/or non-radioactive element in asubject comprising administering a DTPA prodrug and/or composition ofthe present invention to the subject. In some embodiments, a method ofpreventing the incorporation of a radioactive and/or non-radioactiveelement in a subject may comprise administering a DTPA prodrug and/orcomposition of the present invention to the subject prior to thesubject's exposure to the radioactive and/or non-radioactive element.

The term “preventing” and grammatical variants thereof, as used herein,refer to any type of prevention that imparts a benefit to a subject,including avoiding, delaying, and/or reducing the onset and/orprogression of one or more symptom(s) and/or condition(s), reducing theseverity of the onset of one or more symptom(s) and/or condition(s),etc. Those skilled in the art will appreciate that the benefit impartedby the methods of the present invention is not necessarily meant toimply complete prevention (e.g., no detectable incorporation of aradioactive and/or non-radioactive element into a subject's tissue, suchas, for example, bones, and/or organs) and/or abolition of thesymptom(s) and/or condition(s). The symptoms and/or conditions mayinclude those occurring after exposure of the subject to a radioactiveand/or non-radioactive element. In some embodiments, a method ofpreventing the incorporation of a radioactive and/or non-radioactiveelement in a subject comprising administering a DTPA prodrug and/orcomposition of the present invention may prevent, reduce, and/or limitthe amount of the radioactive and/or non-radioactive element that isincorporated in the subject's tissue. Preventing, reducing, and/orlimiting the amount of a radioactive and/or non-radioactive elementincorporated in the subject's tissue may be determined compared to anuntreated subject.

In other embodiments of the present invention, a method of treating asubject prior to exposure to a radioactive and/or non-radioactiveelement is provided comprising administering a DTPA prodrug and/orcomposition of the present invention. In a further aspect of the presentinvention, a method of preventing or reducing the incorporation of aradioactive and/or non-radioactive element in a subject is providedcomprising administering a DTPA prodrug and/or composition of thepresent invention.

“Expose”, “exposure”, and grammatical variants thereof, as used herein,refer to a subject who may come into contact (e.g., a known and/orperceived threat of exposure) and/or has come into contact and/or becomecontaminated with a radioactive and/or non-radioactive element (e.g.,the subject has internalized and/or incorporated a radioactive and/ornon-radioactive element). For example, in some embodiments, a subjectwill be exposed to, has been exposed to, and/or is suspected to beexposed to ionizing radiation (e.g., alpha particles and/or betaparticles) from a radioactive element such that the subject's body mayabsorb about 100 mrem or more of radiation in one year or less. Thus,the subject may receive an absorbed radiation dose of about 100 mrem,500 mrem, 1 rem, 5 rem, 10 rem, 30 rem, 50 rem, 100 rem, 250 rem, 500rem, 1,000 rem or more in one year or less. In some embodiments of thepresent invention, a subject is contaminated with a radioactive element(e.g., the subject has internalized and/or incorporated a radioactiveelement). The exposure to the ionizing radiation may be chronic (e.g.,occurring over a long duration of time such as month(s) and/or one year)and/or acute (e.g., occurring in a short duration of time such asminute(s), hour(s), and/or day(s)).

In some embodiments, a subject will be exposed to, has been exposed to,and/or is suspected to be exposed to a non-radioactive element e.g., aheavy metal, a rare earth metal, etc. A subject may be administeredand/or in contact with a non-radioactive element. For example, in someembodiments, a subject may be administered a gadolinium-based contrastenhancement agent (GBCA), such as a linear and/or macrocyclic GBCA(e.g., before and/or while undergoing a Magnetic Resonance Imagining(MRI) procedure). In some embodiments of the present invention, asubject may be administered a DTPA prodrug of the present inventionprior to exposure to a non-radioactive element, such as, but not limitedto, gadolinium, and/or concurrently with and/or upon exposure to anon-radioactive element. In some embodiments of the present invention, aDTPA prodrug of the present invention is prophylactically administeredto a subject.

Some embodiments include a method of treating a subject to remove anon-radioactive element, such as, but not limited to, gadolinium from asubject and/or a method of preventing the incorporation of anon-radioactive element, such as, but not limited to, gadolinium in asubject The method may comprise administering a DTPA prodrug and/orcomposition of the present invention to the subject. In someembodiments, a method of increasing the removal of the non-radioactiveelement, such as, but not limited to, gadolinium, from the subject maybe provided. A method of the present invention may increase the removalof a non-radioactive element, such as, but not limited to, gadolinium,from a subject compared to an untreated subject. The administering stepmay be before, during, and/or after exposure to the non-radioactiveelement. For example, a DTPA prodrug and/or composition of the presentinvention may be administered to a subject before, during, and/or afterthe subject has received a GBCA. In some embodiments, a method of thepresent invention may treat and/or prevent nephrogenic systemic fibrosis(NSF).

The present invention finds use in both veterinary and medicalapplications. Suitable subjects of the present invention include, butare not limited to avians and mammals. The term “avian” as used hereinincludes, but is not limited to, chickens, ducks, geese, quail, turkeys,pheasants, ratites (e.g., ostrich), parrots, parakeets, macaws,cockatiels, canaries, finches, and birds in ovo. The term “mammal” asused herein includes, but is not limited to, primates (e.g., simians andhumans), non-human primates (e.g., monkeys, baboons, chimpanzees,gorillas), bovines, ovines, caprines, ungulates, porcines, equines,felines, canines, lagomorphs, pinnipeds, rodents (e.g., rats, hamsters,and mice), and mammals in utero. In some embodiments of the presentinvention the subject is a mammal and in certain embodiments the subjectis a human. Human subjects include both males and females of all agesincluding fetal, neonatal, infant, juvenile, adolescent, adult, andgeriatric subjects as well as pregnant subjects.

In particular embodiments of the present invention, the subject is “inneed of” the methods of the present invention, e.g., the subject hasbeen exposed to a radioactive and/or non-radioactive element, it isbelieved that the subject will be exposed to a radioactive and/ornon-radioactive element, and/or it is believed that the subject has beenexposed to a radioactive and/or non-radioactive element. A DTPA prodrug,composition, and/or method of the present invention may be particularlysuitable for children at or younger than about 10 years of age, such aschildren at or younger than about 5 years of age or 1 year of age. Thepresent invention may also be particularly suitable for geriatrics.

The administration step may be carried out prior to, during, and/orafter exposure to a radioactive and/or non-radioactive element or athreat thereof. The administration step may be carried out to deliverone or more doses of a DTPA prodrug and/or composition of the presentinvention, such as 1, 2, 3, 4, 5, 6, 7, 8, or more doses of the DTPAprodrug and/or composition. Exemplary dosage regimens include, but arenot limited to, once a day, twice a day, every other day, once a week,etc. for one or more day(s), week(s), month(s), and/or year(s). Incertain embodiments of the present invention, the administering step iscarried out to remove a radioactive and/or non-radioactive element froma subject. In some embodiments of the present invention, theadministering step is carried out to prevent incorporation of aradioactive and/or non-radioactive element in a subject's tissue and/ororgans and/or to reduce and/or limit the amount of a radioactive and/ornon-radioactive element incorporated in a subject's tissue and/ororgans. In some embodiments of the present invention, the administeringstep is carried out to deliver a therapeutically effective amount ofC2E2. In particular embodiments of the present invention, theadministering step is carried out to deliver a therapeutically effectiveamount and/or prophylactically effective amount of a DTPA di-ethyl esterof Form I and/or Form II to a subject.

As used herein, the term “therapeutically effective amount” refers to anamount of a DTPA prodrug of the present invention that elicits atherapeutically useful response in a subject. Those skilled in the artwill appreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject. Inparticular embodiments of the present invention, a therapeuticallyeffective amount of a DTPA prodrug of the present invention results inthe detectable elimination or removal of a radioactive and/ornon-radioactive element from a subject. Detection of the elimination orremoval of a radioactive and/or non-radioactive element may beaccomplished by measuring the amount of the radioactive and/ornon-radioactive element in the urine, feces, other bodily fluids, and/orexhaled gas from the lungs of the subject. Methods and instruments usedto quantify the amount of a radioactive and/or non-radioactive elementremoved from a subject are known to those skilled in the art andinclude, but are not limited to, quantifying the amount of a radioactiveelement removed using radiation detection equipment such as a gammascintillation counter, a liquid scintillation counter, a flowscintillation analyzer, an alpha spectrometer, a gas proportionalcounter, an ionization chamber, a Geiger-Muller counter, etc. andquantifying the amount of a non-radioactive element removed usingequipment and methods such as inductively coupled plasma massspectrometry, atomic absorption spectroscopy, neutron activationanalysis, X-ray fluorescence, etc.

It is appreciated by those in the field that radioactive elements arevery poisonous and radiotoxic in the body, and that non-radioactiveelements can also be very poisonous and toxic when present in the body.“Remove”, “removing”, “removal”, and grammatical variants thereof, asused herein, refer to removing a portion or all of a radioactive and/ornon-radioactive element from a subject who may become and/or iscontaminated with the radioactive and/or non-radioactive element and mayinclude removing a detectable or nondetectable amount of the radioactiveand/or non-radioactive element from the subject. Removing a portion orall of a radioactive and/or non-radioactive element (including removinga detectable or nondetectable amount of the radioactive and/ornon-radioactive element) from a subject will generally improve themedical condition of the subject. For example, in some embodiments ofthe present invention at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of aradioactive and/or non-radioactive element present in a subject isremoved according to the methods of the present invention.

Methods for quantifying a subject's exposure level to a radioactiveand/or non-radioactive element are known in the art and include, but arenot limited to, quantifying the amount of a radioactive and/ornon-radioactive element in the area to which a subject was exposed,quantifying by estimating how much of a radioactive and/ornon-radioactive element was absorbed or inhaled by a subject,quantifying using whole-body counting instruments, quantifying usingexternal measurements of x-rays emitted from a subject's body,quantifying using physiological based pharmacokinetic models, andquantifying using radioassays of urine, feces, or tissue samples.

According to some embodiments of the present invention, administrationof a DTPA prodrug and/or composition of the present invention to asubject may provide an increase in the amount of a radioactive and/ornon-radioactive element removed from the subject as a whole and/or froma particular tissue and/or organ (e.g., liver, kidney, bone, muscle,etc.) of the subject compared to the corresponding amount of theradioactive and/or non-radioactive element removed from the subject ifthe subject was not administered a treatment to remove the radioactiveand/or non-radioactive element and/or to the corresponding amount of theradioactive and/or non-radioactive element removed from the subject ifthe subject were administered a different treatment and/or differentmode of administration to remove the radioactive and/or non-radioactiveelement (e.g., parenteral administration of DTPA). “Increase”, as usedherein in regard to the amount of removal, refers to an improvement inthe amount of a radioactive and/or non-radioactive element removed byabout 1% or more, such as, but not limited to, about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 150, 200, 250, 300% or more.

The dosage regimen of a DTPA prodrug and/or composition of the presentinvention may be adjusted based on the exposure level and/or thesubject. In some embodiments of the present invention, the amount of aDTPA prodrug of the present invention to be administered to a subjectmay vary according to considerations such as, but not limited to, theparticular polymorphic form of the DTPA prodrug, the dosage unitemployed, the mode of administration, the period of treatment, the ageand/or sex of the patient treated, and/or the nature and extent of thecondition treated.

In some embodiments of the present invention, a DTPA prodrug and/orcomposition of the present invention is designed to deliver about 1 mgto about 2,000 mg of DTPA, a DTPA metabolite, and/or a DTPA prodrug ofthe present invention per kilogram of a subject's total body weight perday, and in some embodiments, about 10 mg to about 1,000 mg, about 1,000mg to about 2,000 mg, about 200 mg to about 600 mg, about 1 mg to about500 mg, about 5 mg to about 250 mg, about 5 mg to about 100 mg, about 15mg to about 45 mg, or about 10 mg to about 40 mg per kilogram of asubject's total body weight per day. In some embodiments, the dose oramount of a DTPA prodrug delivered to a subject may depend on thebinding affinity of the DTPA prodrug for the radioactive and/ornon-radioactive element to be removed from the subject. The duration ofthe administration may be day(s), week(s), month(s), and/or year(s). Incertain embodiments of the present invention, the DTPA prodrug and/orcomposition is administered until there is no detectable amount of aradioactive and/or non-radioactive element present in the subject and/orno detectable amount of a radioactive and/or non-radioactive elementremoved from the subject for a certain period of time.

A DTPA prodrug and/or composition of the present invention may be usedalone or in combination with other therapies and/or therapeutic agents.A DTPA prodrug and/or composition of the present invention may beadministered before, during, and/or after administration of anothertherapy and/or therapeutic agent. In some embodiments of the presentinvention, a DTPA prodrug and/or composition of the present inventionmay be used as a follow-up therapy, such as after parenteraladministration of a chelating agent, such as, but not limited to, DTPA.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES Example 1 Method 1 for Preparing C2E2

The first step in Method 1 for preparing C2E2 (also known as6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid) comprises providing DTPA bis-anhydride. DTPA bis-anhydride iscommercially available and may be obtained from commercial suppliers,such as TCI America of Portland, Oreg., or may be prepared as follows.

Preparation of Bis-anhydride

Step 1—DTPA (3.93 g, 10 mmol) and acetic anhydride (5.72 g, 56.9 mmol)were added to 6.2 mL of pyridine and heated to reflux 65-70° C. for 14hours. The material was filtered through a Buchner funnel and rinsedwith diethyl ether. An off-white powder (DTPA-BA, 1) was collected anddried in a desiccator. ^(1H)NMR (400 MHz, DMSO) δ 3.65 (8H, s), 3.44(2H, d), 2.74 (4H, t), 2.59 (4H, t).

Once DTPA bis-anhydride is obtained, Step 2 is followed to prepare C2E2.

Preparation of C2E2

Step 2—DTPA-BA (1) is used to produce C2E2 (2) by reacting DTPA-BA (1)with ethanol. DTPA-BA (1.02 g, 2.8 mmol), ethanol (0.38 g, 8.2 mmol) andpyridine (0.67 g, 8.4 mmol) were stirred under nitrogen for 24 hours.The product was precipitated in dichloromethane (DCM) (200 mL) at −20°C., filtered while washing with DCM, and dried in a vacuum oven to givean off-white powder (Yield—81.3±13.3, n=4). ^(1H)NMR (400 MHz, DMSO) δ4.07 (4H, q), 3.53 (4H, s), 3.45 (2H, s), 3.43 (4H, s), 2.89 (4H, d),2.84 (4H, d), 1.19 (611, t). Elemental analysis: Predicted—C, 48.10; H,6.95; N, 9.35; 0, 35.60; Actual—C, 47.59; H, 7.22; N, 8.67; 0, 36.29.The synthesis of C2E2 has also been successfully prepared on a 2 gscale.

Method 2 for Preparing C2E2

According to Method 2 for preparing C2E2, C2E2 was manufactured on a 1.5kg scale according to the synthetic scheme in FIG. 1. The synthesis ofbis-anhydride 2 from DTPA (1) is described in European Application No.EP136134A1 (2003), International Publication No. WO 2007/142804 A2, andChemistry, A European Journal, 2004, 10, 3252-3260, the contents of eachof which are incorporated herein by reference in their entirely for theteachings relevant to this paragraph. In each case the penta-acetic acidwas treated with acetic anhydride and pyridine either neat or in thepresence of MeCN and the yield range observed was 95-97%. Thebis-anhydride is then converted to C2E2 by reaction with ethanol. Thisconversion has been used to prepare 800 g as well as 1.5 kg of C2E2.

Preparation of Bis-anhydride (2):

Diethylenetriamine pentaacetic acid (2029 g, 5.16 mole) was dissolved inacetonitrile (1100 mL) with agitation. Acetic anhydride (1450 mL, 15.3mole) and pyridine (1660 ml, 20.5 mole) were added and the reaction washeated to 60° C. for 4 hours. The reaction mixture was cooled to 22° C.and t-butylmethylether (MTBE) (800 mL) was added. The reaction mixturewas filtered and the solid obtained was washed with MTBE (3000 mL). Thesolid was dried in a vacuum oven at 40° C. Yield: 1792 g, 97%.

Preparation of C2E2:

The bis-anhydride (1792 g, 5.02 mole) was slurried in absolute ethanol(9000 mL) and heated to reflux with agitation for 1.5 hour. The heatedreaction mixture was filtered through Celite and cooled below 20° C. Theresulting slurry was filtered and washed with cold ethanol (2800 mL)followed by methyl tert-butyl ether (MTBE) (3500 mL). The Celite cakewas slurried in ethanol (800 mL) and heated to 70° C. and filtered. Oncooling, the slurry obtained was filtered and washed with ethanol andMTBE. The combined isolated solids were dried in a vacuum oven at roomtemperature. Yield: 1488 g, 66%.

The Certificate of Analysis for the 1.5 kg lot of C2E2 is providedbelow. Since C2E2 has no chromophore, a reverse phase HPLC method witheither evaporative light scattering detection (ELSD) or Charged AerosolDetection (CAD) was developed and qualified for analysis of C2E2 andimpurities.

Certificate of Analysis for C2E2

Structure:

-   -   Material: C2E2    -   Lot No: 020WM050    -   Batch Size: 1.72 kg    -   Appearance: A white solid.    -   Identity: Determined by ¹H NMR. Data are consistent with the        structure.    -   Chromatographic Purity: 97.5% (area %) by Reversed-Phase        HPLC-ELSD    -   Unspecified Individual impurities (area %, average of        duplicates):    -   RRT0.33 0.33%    -   RRT1.08 1.39%    -   RRT1.28 0.77%    -   Total Impurities: 2.49%    -   Moisture by KF: 0.17%    -   DSC: Single endotherm centered at 141.6° C. observed.        Polymorphs:

The possibility of multiple C2E2 polymorphs arose during the analysis ofC2E2 dosing solutions. The C2E2 dosing solutions were prepared with C2E2of Lot 050 obtained using Method 2. Investigating polymorphs isimportant as it affects the physiochemical properties of the bulkpowder. Metastable crystalline solids have high chemical potential andare unstable. The difference in physiochemical properties, particle sizeand surface area can alter the bioavailability of the drug due tochanges in dissolution rate. The goal of the following studies was toidentify the most stable polymorphic form as any other polymorphs aremetastable and could change during storage. This phase change couldresult in precipitation from solution or instability during storage.

Analysis of Dosing Solutions:

Samples from dosing were received for analysis and checked for leaks andlabeling before storing at 4° C. The formulation samples (0, 20, 60, and100 mg/mL) were diluted to an expected concentration of 0.40 mg/mL andanalyzed by HPLC-CAD.

During preparation for analysis, the 60 and 100 mg/mL solutions hadnoticeable solid precipitated from the formulations, as seen in FIG. 2.The diluted samples were prepared and analyzed as if the solutionscontained the indicated amount of C2E2. The results shown below in Table1, show poor percent recoveries with no clear trends related toconcentration. Both 20 and 100 mg/mL solutions fall outside the acceptedrange of 85-115% recovery. The 60 mg/mL solution is technically withinthe recommended preparation range, but the formulation contained solidprecipitate.

TABLE 1 Percent recovery of C2E2 from dosing formulations. Dosingformulation Percent recovery (%) concentration (mg/mL) of C2E2 0 0 20184.87 60 92.08 100 220.42Stability Study in Water:

Method 2 was used to prepare the C2E2 for the dosing formulations.Specifically, Lots 050 and 005-2 were prepared using Method 2, whereasthe Reference standard and MTD Lot were prepared using Method 1. For thedosing formulations, de-ionized water (DI water tap in room 18/19) wasused with the C2E2 prepared using Method 2. The dosing formulations werecompared with C2E2 formulations prepared using Milli-Q water. The pH ofeach sample was recorded and the values are listed below in Table 2.

TABLE 2 pH values of solubility samples. pH of formulations pH offormulations prepared using de- prepared using Formulation ionized waterMilli-Q water Vehicle (water) 6.97 4.95 20 mg/mL (Lot 050) 3.04 60 mg/mL(Lot 050) 2.93 100 mg/mL (Lot 050) 2.93 2.60 100 mg/mL (Lot 005-2) 2.54100 mg/mL Purified C2E2 2.43 (Reference std) 100 mg/mL (MTD Lot) 2.35

The pH of the vehicles differs by nearly two units. The pH of thedifferent 100 mg/mL preparations of Batch 050 differs by only 0.3 pHunits, though the concentrations are likely not equivalent due theamount of precipitated material in the sample. The pH values of thedifferent C2E2 preparations made using Method 1 range from 2.35 to 2.60.While these differences are small, it does seem likely that the C2E2 inLots 050 and 005-2 have a slightly higher solution pH than that of theMTD Lot and Reference standard (the Reference standard is a purifiedC2E2 from the MTD Lot). While not wishing to be bound to any particulartheory, this may be due to the presence of impurities in the Referencestandard and MTD Lot.

In order to re-create the unusual solutions, 1-mL samples of C2E2 (Lots005-2 and 050; MTD Lot; and Reference std) were made at concentrationsof 100 mg/mL in Milli-Q water. A set of samples was stored at roomtemperature, in the refrigerator (4° C.), and in the −20° C. freezer.Observations of these solutions at various time points show that within24 hours the reference material starts to precipitate as a fine powder,regardless of the storage temperature. Lot 050 also begins to come outof solution after three days, but as slightly clumpy materials. Storageat 4° C. exacerbates the precipitation of Lot 050. After 8 days, boththe Reference Standard and Lot 050 have significant amounts of soliddetected at 4° C. and room temperature and crystals have started toform. Freezing and thawing does not cause unreasonable precipitation.The two preparations that are relatively less pure (the MTD Lot and Lot005-2) do not come out of solution. While not wishing to be bound to anyparticular theory, this is likely because more impurities increase thesolubility.

Differential Scanning Calorimetry (DSC):

Samples of C2E2 (˜5 mg) were crimped and evaluated by DSC over a rangeof 25° C. to 320° C. with a heating rate of 10.00° C./min. A sample wasalso cycled three times from 25 to 150° C. with a heating rate of 10°C./min and a cooling rate of 5° C./min. The results of DSC analysis areshown in FIG. 3. Both the MTD Lot and the Reference Standard show amelting point near 120° C. Lot 050 has a melting point of about 140° C.The material filtered from the dosing formulation for Lot 050 also has amelting point of about 140° C., suggesting that the C2E2 used for dosingpreparations has the same composition as that which comes out ofsolution. The differences in melting point suggest the presence ofpolymorphs possibly due to the presence of different solvates.

Interestingly, the impure batch of C2E2 (Lot 005-2) has two meltingpoints, one near 120° C. and the other near 140° C. While initially itwas assumed that the second peak was due to C2E3 detected by MS andHPLC, without wishing to be bound to any particular theory, it now seemslikely that the two peaks are indicative of two types of C2E2 present.Both of these peaks are broadened, probably a result of impurities (suchas the C2E3 detected by NMR/HPLC/MS).

C2E2 starts to combust at temperatures above 150° C. When the mixedbatch of material (Lot 005-2) with two melting points is cycled slowlyfrom 25 to 150° C., no shift from one melting point to another is seen.In fact, after one heating cycle, no other peaks were seen, though nocombustion was seen on the endotherm. While not wishing to be bound toany particular theory, it had been anticipated that a shift from thelower temperature to the higher would occur, but it is likely that thecycling conditions were not ideal.

Competitive Ripening Experiment:

A brief competitive ripening experiment was conducted to determine themore stable melting point. 50 mg each of Lot 050 and the Referencestandard were combined in 500 μL of water and stirred at roomtemperature overnight. After stirring, the remaining solid was filteredfrom the liquid, allowed to dry and analyzed by DSC. The competitiveripening experiment showed that the solid material recovered from astirred mixture of two materials had only a single melting point of 140°C. While not wishing to be bound to any particular theory, the favoringof the higher temperature suggests that the material with the highermelting point is the more stable compound.

Thermogravimetric Analysis:

A second thermal analysis method, thermogravimetric analysis (TGA) wasused to characterize the compounds. TGA measures the thermally inducedweight loss as a function of temperature and thus can be used to studythe desolvation or decomposition of the compound. This information isuseful in combination with the DSC data, as desolvation processes areaccompanied by a weight change, whereas solid-liquid or solid-solidphase transitions are not associated with a change and therefore notpresent on the TGA thermogram. Solids from both synthesis methods wereplaced on the TGA and heated using the following method. Initialtemperature was set to 25° C. and the temperature was increased at 10°C./min to 100° C. An isothermal step was introduced at 100° C. for 5 minto ensure all water loss had occurred, and then the temperature wasfurther increased at 10° C./min to 170° C. (FIG. 4).

Firstly, the TGA data confirms that the endotherms observed on the DSCare melting points as there is no significant weight change observed onthe TGA thermogram for either compound at the associated temperature,which is indicative of a phase change rather than decomposition ordesolvation. Lot 050 exhibits a gradual weight loss that may be due tothe presence of surface water. The MTD Lot has no significant loss inweight aside from a 0.5% decrease between 116-123° C. While not wishingto be bound to any particular theory, due to the fact that there is nostoichiometric weight loss seen, it is unlikely that this is caused bythe presence of a hydrate or solvate. The small weight loss may be animpurity that is released as the polymorph begins to melt. Bothpolymorphs begin to decompose around 170° C. as seen by the rapid weightloss.

Elemental Analysis:

Elemental analysis was performed on the Reference Standard and Lot 050samples. The elements requested for analysis were C, H, N, and O. Theresults of the elemental analysis C2E2 are shown in Table 3. TheReference Standard sums to 98.92% of the total weight, indicating a verysmall level of contamination. On the other hand, the Lot 050 sample has1.93 weight percent unaccounted for. Thus, it is unlikely for solvent toaccount for the difference seen between synthetic lots.

TABLE 3 Percent composition of C2E2 preparations. Percent PercentPercent Percent Carbon of Hydrogen of Nitrogen of Oxygen of Sample ID wt% theoretical wt % theoretical wt % theoretical wt % theoretical UNCRef. 48.1 100 6.57 94.5 9.40 100.53 34.85 97.89 std Lot 050 48.3 100.426.60 94.96 9.31 99.57 33.86 95.11 Theoretical 48.10 6.95 9.35 35.60IPC/MS:

To eliminate a metal chelate as a possibility for the differences in theC2E2, each preparation was analyzed by ICP-MS. Dilutions of C2E2(Reference Standard and Lot 050) were made in water to a concentrationof 110 mg/mL. The results are shown in FIG. 5. There are no differencesof note between the two C2E2 samples. Sodium has the highestconcentration detected, between 2 and 3 μg/mL for the 110 mg/mL C2E2.Other elements (magnesium, potassium, iron and zinc) are present atconcentrations less than 500 ng/mL, and are nearly equal between the twosamples. This small amount of metal is possibly due to the water orglassware used and is unlikely to be responsible for the distinctdifferences in the melting points seen on the DSC.

X-Ray Powder Diffraction:

Samples of C2E2 (Reference Standard, MTD Lot, Lot 050 and Lot 005-2)were analyzed by X-ray powder diffraction by K Sueda at GSK. Analysis ofC2E2 samples was conducted by K Sueda and results are shown in FIGS.6-8. All four spectra are shown in FIG. 6. Overlays of the ReferenceStandard and MTD Lot show the same crystal structure with only minordifferences (FIG. 7) and are distinctly different from the crystalstructure of Lots 050 and 005-2. Lots 050 and 005-2 are different fromeach other (confirming the HPLC and DSC data) (FIG. 8). Lot 005-2 moreclosely resembles the Reference Standard and MTD Lot and, withoutwishing to be bound to any particular theory, may be a mixture ofcrystals.

While not wishing to be bound to any particular theory, the differencesbetween the C2E2 samples is not due to solvation or chelation, and ismost likely a case of polymorphism. It appears that Lot 050 is morestable (higher melting point) in its solid state.

Scanning Electron Microscopy:

Final confirmation of the presence of two polymorphs was obtained usingscanning electron microscopy. Powdered samples were sputter coated witha 5 nm thick coating and imaged. The C2E2 MTD Lot is scaly in nature, asopposed to the divergent habit of Lot 050, which possesses a distinctcrystal form as shown in FIG. 9.

While not wishing to be bound to any particular theory, the datadiscussed above suggest that C2E2 has at least two polymorphic forms.The higher melting point and lower solubility (observed as precipitatingout during storage in the refrigerator) suggests that the polymorph seenin Lot 050 is the most stable.

Recrystallization Studies of C2E2:

Lot 050 has known impurities of ethanol and API related material, likelysmall amounts of C2E3 and C2E1. The chromatogram of this material isshown in FIG. 10. The small peak with RT of 11.86 min has an approximatearea of 5.2% in this injection. There is no C2E1 detected (RT ˜4.5 min),and the peak at 2.5 min is consistent with the injection dead volume.

A solution of C2E2 (100 mg/mL) was prepared in water and upon sittingfor several weeks produced clear cubic crystals. A small amount of thesecrystals were dissolved in water and analyzed by HPLC. The resultingchromatogram is shown in FIG. 11. The peak area of C2E3 (RT 11.8)decreased slightly to ˜2.6%, but the C2E1 peak has increased to 5.7%.

Recrystallization of C2E2 from ethanol was also identified as a possibleroute to improve purity. As such, small portions of C2E2 were heated inethanol (200 proof) until all material was dissolved. The vials werecooled and C2E2 precipitated slowly. The material resulting from ethanolheating and cooling was white and powdery in nature, microcrystallinerather than discrete visible crystals. FIGS. 12 and 13 showchromatograms of the solid and liquid material from a one hour heatingin ethanol (dissolution of C2E2 in ethanol is slow). The solid materialrecovered from this experiment shows increased percentages of C2E3(8.8%) and C2E1 (4.3%), suggesting that this route of ‘purification’ isunlikely to result in a reference standard quality material. Thefiltrate, while enriched in C2E3 (RT 11.8) also contains a significantamount of C2E2. This residual material suggests that a significantamount of C2E2 would be sacrificed while not generating C2E2 in thedesired purity.

While not wishing to be bound to any particular theory, this study showsthat the recrystallization of C2E2, while seemingly simple, is likely tointroduce larger amounts of additional impurities while decreasing theC2E3 present. While C2E2 is relatively stable in solution, the additionof heat or moisture leads to esterification or de-esterification and islikely to produce C2E3 or C2E1 and DTPA.

Aqueous Solubility

Preliminary Test:

A small sample of C2E2 (0.1 g, JH012B) was weighed out and placed in a25 mL graduated cylinder. Increasing volumes of distilled water wereadded according to the chart below:

Volume water 0.1 0.5 1 2 10 100 >100 added (mL) Approximate >1000200-1000 100-200 50-100 10-50 1-10 <1 solubility (g/L)

After each addition of the indicated amount of water, the mixture wasstirred for 10 minutes and visually checked for any dissolved parts ofthe sample. The preliminary test showed that C2E2 was soluble in 10 mLof water after 10 minutes. The approximate solubility is therefore 10-50g/L.

ADME

Metabolic Stability:

Caco-2 cells are a human colon epithelial cancer cell line thatexpresses transporters, efflux proteins and phase II enzymes and can beused to correlate cell permeability with drug absorption. In addition todetermining the apparent permeability coefficient, an estimation of C2E2metabolism during transport can be obtained. The aim of thesepreliminary studies was to evaluate a range of C2E2 concentrations (25,50 & 100 μM) in order to determine a linear concentration range in whichfurther studies could be performed.

Caco-2 cells (passage #39) were cultured in 75 cm²T-flasks at 37° C. ina 5% CO₂ constant humidity environment. The medium was replaced 3 timesa week and the monolayers were sub-cultured to passage #41. When platingthe cells 0.5 mL of a 120,000 cells/mL suspension was added to eachapical (AP) compartment of the first and then 1.5 mL of cell culturemedium was placed into the basolateral (BL) compartment of theTranswells®. Following 21-24 days on the Transwell® plates with mediachanges 2-3 times a week the Caco-2 transport studies were performed.

Prior to the transport experiments the transport medium was warmed to37° C. Transport medium consisted of Hank's Balanced Salt Solution(HBSS) (1×) with calcium and magnesium 500 mL with the addition of 1MHEPES buffer (5 mL) and 1.25M D-Glucose solution in HBSS (10 mL). Thecell culture medium was removed from the Transwells® to be used for theexperiment and each well was washed twice with warm transport medium.The Transwells® were then pre-incubated for 30 min with transportmedium. Finally, the trans-epithelial resistance (TEER) values for eachTranswell® were measured using a voltohmmmeter and chopstick electrode.The TEER values should be in the range of 300-750 and should vary aslittle as possible. Dosing solutions were prepared in warm (37° C.)transport medium. Pre-incubated transport medium was removed from theTranswells® and 1.5 mL fresh 37° C. transport medium was pipetted intothe acceptor compartment. At time zero 0.5 mL of the dose was pipettedinto the donor compartment. The Transwells® were then placed in a 37° C.incubator for the duration of the experiment. At each time point (20,40, 60 & 120 min) the transport medium was collected and diluted withacetonitrile. The Transwell® was transferred into a new acceptorcompartment containing 1.5 mL of fresh pre-heated transport medium. Theconcentration of C2E2 was then determined by LC-MS/MS. The concentrationof the dosing solutions was also measured to determine the concentrationin the donor compartment at time zero. During these preliminary studies,the transport from the apical to the basolateral compartment wasinvestigated.

C2E2 was detected in the basolateral receiver compartment at allconcentrations tested. The percentage of dose that passed through themembrane was similar for all three concentrations as shown in FIG. 14.Between 18-22% of the dose was detected in the receiving compartment.Although none of the lines are statistically different, the 100 μMplates exhibited the lowest percentage transport.

One of the goals of these experiments was to determine if at these dosesthere was a linear response. FIG. 15 shows the flux calculated for eachconcentration. As the flux is a measure of the mass of material movingthrough the membrane per unit time if the dose response was linear thenhigher concentrations should exhibit a higher flux. In these experimentsthe highest dose level had the lowest flux. While not wishing to bebound to any particular theory, this could be because the concentrationis no longer in the linear range; alternatively it may be due to theprocessing of this data on the mass spectrometer as the 100 μM sampleswere run in a separate run. The data from all the sample concentrationsshould be combined and reprocessed to ensure that the data was processedidentically.

FIG. 16 shows a decrease in the apparent permeability coefficient withincreasing dose of C2E2. If the C2E2 was absorbed by passive diffusionthen the permeability coefficient should be constant. Thus the decreasemay be due to a number of reasons. The absorption may be transportmediated and at increasing concentrations of C2E2 either apical uptakeinto the Caco-2 cell or basolateral efflux becomes saturated.Alternatively p-gp may efflux the C2E2 on the apical membrane therebylimiting absorption. Additional experiments such as measuring thetransport of C2E2 from the basolateral to the apical side and assessingthe effect of p-gp inhibitors on the transport of C2E2 are required todetermine the cause of the decrease in P_(app) with increasingconcentration.

The relationship between the apparent permeation coefficient determinedin Caco-2 experiments with the percentage dose absorbed in humans isdescribed in Shiyin Yee (Pharm. Res., 1997 June; 14(6):763-6.), thecontents of which is incorporated herein by reference in its entiretyfor the teachings relevant to this paragraph. Based on the data ofShiyin Yee, the observed apparent permeation coefficient of 4.88×10⁻⁶cm/sec suggests that C2E2 would be moderately (20-70%) absorbed inhumans providing that dissolution and GI metabolism are not limitingfactors.

Efficacy

Summary of C2E2 Single Dose Decorporation in Rats:

This study was to assess the efficacy of a single oral gavage dose ofC2E2 in ²⁴¹Am contaminated rats. A 200 μL aliquot was withdrawn from astock solution of ²⁴¹Am nitrate (0.1 mCi in 5 mL 1 M HNO₃), evaporatedto dryness, dissolved in concentrated HNO₃ (15 M, 5 mL) then evaporatedto dryness and dissolved in dilute HNO₃ (0.1 M, 8 mL) to form theinjection solution. Groups of 4 female Sprague-Dawley rats werecontaminated with 250 nCi of ²⁴¹Am nitrate by IM injection (0.1 μL) thenwere administered single doses of C2E2 by gavage at 200, 600, or 1000mg/kg under ad lib feeding conditions utilizing a 10% w/w solution insterile water (dose volumes were 2, 6, and 10 mL/kg, for the 200, 600,and 1000 mg/kg doses, respectively). For the fasted component of thestudy, the animals were fasted 24 hours before treatment then for 2hours post drug administration. Control groups with ad lib access tofood and water were either untreated or administered an IV dose of thecalcium salt of DTPA (Ca-DTPA) at 14 mg/kg (target dose volume of 0.7mL/kg and concentration of 20 mg/mL). Treatment was administered 1 dayafter contamination. All animals were euthanized 7 days aftercontamination and the following tissues were collected: liver, bothkidneys, both femurs as well as the ipsilateral and the contralateralhind leg muscle tissue and the pelt from the injection site. Urine andfeces were collected from the time of contamination through euthanasia.Cage washings were also collected at the end of the study and consideredpart of total urine collection. Two aliquots (100 μL) of the injectionsolution were removed and ²⁴¹Am gamma activity was determined usingradiomatic detection. The average of these standards is the injecteddose used for all animals. All tissues and samples were counted in asimilar manner. Americium content was expressed as a percentage of theinjected dose. Overall recovery of radioactivity across all groups inthis study was approximately 90%. Table 4 shows total decorporation andresidual liver burden at euthanasia in untreated controls and after asingle dose of Ca-DTPA or C2E2. The primary route by which both DTPA andC2E2 enhanced elimination of ²⁴¹Am occurred was by urinary clearance.Results suggest feeding state did not affect total ²⁴¹Am clearance.Fecal clearance of ²⁴¹Am was not altered by feeding status, anobservation that is consistent with the similar liver burden between thetwo groups at the end of the study. This study demonstrated C2E2increased total body decorporation in female rats and reduced ²⁴¹Amliver burden in a dose-dependent manner. As with DTPA, enhanceddecorporation by C2E2 occurred through enhanced urinary clearance.

TABLE 4 Americium Decorporation 7 days After IM Contamination: Treatmentwith a Single Dose of C2E2 24 h after Contamination. Total Cage WashDecorporation Liver burden Urine (%) Feces (%) (%) (% injected) (%injected) DTPA Dose (mg/kg)  14 22.1 ± 2.6 10.3 ± 1.7  3.6 ± 0.8 36.1 ±4.6 12.3 ± 1.2 C2E2 Dose (mg/kg)  0  4.7 ± 1.0 4.9 ± 1.0 1.6 ± 0.5 11.2± 2.1 21.4 ± 2.5 200  6.7 ± 1.5 5.9 ± 0.8 1.8 ± 0.5 14.4 ± 0.7 17.7 ±2.1 200 (fasted)  8.4 ± 1.3 4.7 ± 2.1 1.3 ± 0.5 14.3 ± 3.2 20.9 ± 3.5600 12.4 ± 1.3 7.7 ± 2.0 2.2 ± 0.8 22.3 ± 3.1 18.6 ± 4.5 600 (fasted)14.3 ± 2.9 7.3 ± 1.5 2.6 ± 0.8 24.2 ± 3.3 18.9 ± 2.3 1000  13.5 ± 1.76.9 ± 1.7 3.4 ± 0.3 23.8 ± 3.0 14.5 ± 1.5 1000 (fasted)  19.8 ± 3.9 7.1± 1.3 4.5 ± 0.7 31.3 ± 5.5 13.7 ± 3.1The Full Details of this Study are as Follows:

Male and female Sprague Dawley rats (n=4/gender/dose) were administeredsingle oral doses of diethylenetriaminepentaacetic acid diethyl ester(C2E2) by gavage 24 hours after intramuscular contamination with²⁴¹Am(NO₃)₃ (250 nCi) under ad libitum and fasted conditions. Animalsreceived single oral nominal doses of 200, 600, or 1000 mg/kg C2E2.Untreated animals and animals treated with an intravenous dose ofCa-DTPA (13.3 mg/kg) 24 hours after contamination served as controls;only ad libitum conditions were used for control animals. Totalradionuclide decorporation and radionuclide tissue burden were evaluatedseven days after contamination.

C2E2-dose dependant americium decorporation was observed in both maleand female rats following C2E2 treatment, with benefits of C2E2treatment evident at doses of 600 mg/kg and above. At 1000 mg/kg, asingle C2E2 oral dose is comparable, in terms of efficacy, to anintravenous dose of Ca-DTPA (13.3 mg/kg), although equivalence analysishas not been conducted. Of the other effects examined, the studyestablished that rat gender significantly influences americiumdecorporation and tissue burden, but fasting prior to administration ofthe C2E2 dose does not. This determination resulted in data from adlibitum and fasted groups being combined for statistical analysis; maleand female groups still required separate analysis. Consistent with aprevious single dose toxicology study (UNC-11.240-RMTD), no C2E2-relatedclinical signs were observed over the 7-day period.

By reducing the amount of americium in key tissues and increasing theamount of americium that is removed from the body, a single oral C2E2dose can effectively treat americium contamination in a rat wound modelof contamination. C2E2 efficacy has been demonstrated at dosesconsiderably below the maximum tolerated single dose (2000 mg/kg) forthe species.

Introduction and Objective:

The objective of this study was to investigate effects of food, gender,and dose, on the tissue burden and decorpoartion of ²⁴¹Am following asingle oral dose of C2E2. The delay between contamination and treatmentselected for this study was 24 hours; this reflects a realistictreatment delay for an orally administered drug in a mass casualtysituation. Results obtained from this study will be used to design doseregimen pharmacology studies in rats.

Materials and Methods:

This study was conducted under The University of North Carolina atChapel Hill Protocol UNC-10086-E7SD.

Test and Control Article:

The Test Article, C2E2 from the MTD Lot, was documented to be 98% pure,no correction for purity was made in preparing dose solutions. The TestArticle was stored at room temperature. Control Article (Ca-DTPA; HamelnPharmaceuticals) was stored at room temperature. The records ofdisposition are maintained in the study file.

Test and Control Article Formulation:

The Test Article was dissolved on each day of dosing in sterile water(Hospira Inc.) to achieve a 10% w/w solution. Control article (Ca-DTPA)was diluted in 0.9% Saline (Baxter International) on the day of dosing.Analysis of dosing solutions was not performed.

Radionuclide Preparation:

The ²⁴¹Am injection solution was prepared. A 200 μL aliquot of the stock²⁴¹Am(NO₃)₃ solution (0.1 mCi/mL in 1 M HNO₃) was removed and placed ina 20-mL glass vial. The aliquot was evaporated to dryness in a stirredoil bath. Concentrated nitric acid (15 M, ≈5 mL) was added to the dryamericium nitrate and the resultant solution evaporated to dryness. Toform the injection solution, dilute nitric acid (0.1 M, 8 mL) was addedto the dry 20-mL vial, the solution was then filtered into a 10 mLinjection vial, capped and stored securely until use.

Test System:

Naive and jugular catheterized male and female Sprague Dawley rats werepurchased from Charles River Laboratories (Raleigh, N.C.). Shortly aftertheir arrival at the laboratory, the animals were removed from theshipping cartons and examined. No evidence of disease or physicalabnormalities was identified. At contamination, the animals were 260-310g male and 210-320 g female. The fate of all animals is documented inthe study records.

Allocation:

Body weights were measured prior to ²⁴¹Am administration. Individualweights of animals placed on study were within ±20% of the mean weightfor each gender with one exception (female with body weight −20.5% ofthe mean). Animals considered suitable for the study based on thepre-study observations were assigned to test or control article groups.Animals were not randomized due to space limitations.

Identification:

Each animal was assigned a temporary identification number and cagelocation upon receipt. Immediately after contamination, animals wereassigned a cage number and location and a unique number assigned by theTesting Facility (Table 5).

Animal Husbandry:

Animals were individually housed in metabolic cages (Techniplast USA).Environmental parameters were recorded and are included in the studyfile. Temperature (desired range of 68±3° F.), humidity (approximatelyof 30 to 70%), lighting (12 hour light/dark cycle; 0700-1900 hrs), andair exchanges (20-30 air changes per hour) in the housing area weremonitored to ensure that the conditions were maintained to the maximumextent possible. Room sanitation and opening/closing room door may havetemporarily created excursions. Drinking water from the Chapel Hill,N.C. municipal water system and standard rodent chow (Labdiet, ProlabRMH 3000) were provided ad libitum except where indicated.

Experimental Groups:

Individual animal number assignments and experimental groups are shownin Table 5.

TABLE 5 Animal Number Assignments. Animal Identification Number DoseUNC-10086-E7SD (mg/kg) Food Access Male Female Untreated ad libitum447-450 443-446 200 ad libitum 475-478 431-434 600 ad libitum 459, 460,462, 492 487-490 1000  ad libitum 465, 466, 496, 497 491-494 14(i.v.Ca-DTPA) ad libitum 471-474 467-470 200 Fasted 479-482 483-486 600Fasted 451-454 435-438 1000  Fasted 455-458 439-442 Total 32 32Americium Contamination:

Male and female Sprague Dawley rats were contaminated with ²⁴¹Am(NO₃)₃(0.25 μCi) by intramuscular injection (0.1 mL, 0.1 M HNO₃) into theright hind leg, under isoflurane anesthesia. Immediately afterinjection, body weights were recorded (Day 0 weight) and animals wereplaced in individual metabolic cages.

Fasting:

Approximately eight hours after contamination—and 16 hours before testarticle administration—rats assigned to the Fasted groups had access tofood restricted by removing the food chamber from their metabolic cagesand collecting all loose chow from their housing chambers. Food chamberswere replaced 1 hour after oral gavage treatment, restoring access tofood. At all other times all animals had ad libitum access to food.

Administration of Test Article:

Male and female Sprague-Dawley rats assigned to test groups eachreceived a single oral gavage dose of 0, 200, 600, or 1000 mg/kg of C2E2utilizing a 10% w/w solution in water 24 hours after contamination. Insome animals, brief exposure isoflurane (5% by vaporizer) was requiredduring dosing to reduce the spread of radionuclide. Dose volume in thesegroups ranged from 2-10 mL/kg. Doses were based upon the Day 0 weight(immediately after contamination). Mass of the dosing solutionadministered to each rat was recorded.

Administration of Control Article:

Under isoflurane anesthesia four male and four female Sprague-Dawleyrats each received a single intravenous dose of the Ca-DTPA solution(13.3 mg/kg) via a jugular vein catheter 24 hours after contaminationutilizing a 0.67 mL/kg followed by a 0.2 mL flush of 0.9% sterilesaline. The mass of the administered Ca-DTPA dosing solution wasrecorded. Doses were based upon the Day 0 weight (recorded immediatelyafter contamination).

Euthanasia:

Scheduled euthanasia by isoflurane overdose and thoracotomy wasperformed seven days after contamination.

Viability Checks:

All animals were observed at least once daily for morbidity, mortality,and general appearance.

Excreta:

Excreted urine and feces were collected daily, allowed to dry overnight,transferred to 20-mL scintillation vials, weighed and placed in a gammacounter (Wizard2 2480, Perkin Elmer) for detection of ²⁴¹Am gammaactivity.

Body Weights:

Each rat was weighed at contamination (Day 0) and the terminal bodyweight (Day 7) was taken at necropsy.

Food Consumption:

Food consumption was monitored daily by visual inspection to be surethere was a sufficient amount of feed. The animals had a continuoussupply of feed available to them except where indicated.

Post Mortem:

Scheduled animals were brought to the necropsy room, euthanized byisoflurane overdose followed by thoracotomy, their terminal body weightswere recorded and selected tissues removed, weighed and placed in agamma counter (Wizard² 2480, Perkin Elmer) for detection of ²⁴¹Am gammaactivity.

Sample Analysis:

The total amount of ²⁴¹Am administered to the animals was determined byquantifying the 59.7 keV photons emitted by ²⁴¹Am in duplicate aliquots(100 μL) of the injection solution using a gamma counter (wizard series,Perkin-Elmer). A counting window from 40-80 keV and a 60-second countingtime were used for acquisition and each reading was corrected forbackground at acquisition. All experimental tissues and samples werecounted using the same gamma counter and protocol. For all the samples,²⁴¹Am content was expressed as a percentage of the initial dose. Thefemur from the leg opposite to the injection site was scaled by a factorof 20 to estimate total skeletal ²⁴¹Am burden.

Statistics:

Statistical analysis was performed for final decorporationparameters—total decorporation, liver burden, wound retention andestimated skeletal burden. All animals treated with C2E2 were analyzedby ANOVA to evaluate the effects of gender, dose and fed state.Non-contributing effects were removed from the model and control data(untreated and Ca-DTPA treatment groups) were included for subsequentanalysis. ANOVA was performed and Least squares means calculated for allthe groups in this model. Comparison of means was made using theTukey-Kramer adjustment for multiple comparisons, with p<0.05 consideredsignificant.

Results

Animals:

Contamination and subsequent treatment of male and female rats occurredin nine cohorts between June 9^(th) and August 24^(th). Three male ratsin a cohort started August 2^(nd) died as a result of experimenter errorduring gavage treatment, additional animals were added to the August24^(th) cohort to replace these lost animals. Some difficulty wasexperienced in dosing female rats. Specifically, the dosing solution wasobserved in the mouth during gavage in five female rats. In the twoinstances where dosing solution remained confined to the mouth, theobserved volume was considered insignificant with no adverse effectsnoted; these rats were retained in the analysis. However, in three ratssome loss of dose from the mouth occurred and partial aspiration of thedose may have occurred as brief difficulty breathing was observed.Although no adverse effects of dosing in these animals were observed atone hour after dosing and they completed the in-life portion of thestudy these animals were excluded from data analysis.

Clinical Observations:

No adverse C2E2 exposure-related clinical observations for male rats orfemale rats were identified in any of the dose groups. Individual animalclinical observation data are included with daily excreta data on DailyCage Observations sheets in the study file.

Body Weights:

Body weights were recorded at contamination and at euthanasia for allanimals. Mean body weights and percentage change in body weight duringthe study are shown in Table 6 and Table 7. Although significantdifferences are observed between different groups in both male andfemale animals, these are not anticipated to influence the outcomes ofthe study. One female animal (body weight −20.5% of mean) was includedin the study despite being outside the ideal weight range (20% of meanweight for gender).

TABLE 6 Mean Body Weights of Male and Female Sprague-Dawley Rats C2E2Treatment ad libitum Fasted (mg/kg) Day 0 Day 7¹ Day 0 Day 7¹ MaleUntreated 276.3 ± 8.5 310.2 ± 19.4 ND ND 200 296.3 ± 9.4 326.5 ± 25.7291.3 ± 12.7 314.2 ± 14.4 600 292.8 ± 5.1 322.2 ± 6.4 275.9 ± 9.7 310.6± 9.1 1000  290.5 ± 6.6 321.2 ± 6.5 270.3 ± 5.8 302.8 ± 14.2  14 301.0 ±4.0 314.6 ± 20.4 ND ND (iv Ca-DTPA) Female Untreated 294.8 ± 11.4 279.3± 19.1 ND ND 200 276.8 ± 17.3 276.5 ± 15.1 245.0 ± 11.6 246.8 ± 9.6 600247.0 ± 13.8 249.7 ± 14.5 280.5 ± 28.0 273.5 ± 19.2 1000  232.5 ± 19.3234.2 ± 21.4 276.3 ± 17.9 262.7 ± 23.8  14 273.6 ± 7.7 273.3 ± 11.2 NDND (iv Ca-DTPA) ¹Mean body weights are terminal weights

TABLE 7 Mean Percentage Body Weight Change. C2E2 Treatment Male Female(mg/kg) ad libitum Fasted ad libitum Fasted Untreated 12.4 ± 7.8 ND −5.3± 3.1  ND 200  10.4 ± 11.0  8.1 ± 8.4 0.0 ± 2.5  0.8 ± 2.2 600 10.1 ±3.1 12.6 ± 3.1 1.1 ± 1.2 −2.3 ± 3.3 1000  10.6 ± 0.8 12.0 ± 4.8 0.7 ±2.1 −5.0 ± 2.9  14  4.5 ± 6.1 ND −0.1 ± 1.9  ND (iv Ca-DTPA)Data Analysis of ²⁴¹Am Excretion:

Sixty-four rats completed the study, of these 61 rats (32 male and 29female) were included in data analysis. Three fasted female rats (1×600mg/kg; 2×1000 mg/kg) were excluded from the dataset as they did notreceive a complete dose.

Effect of Food on ²⁴1 Am Decorporation:

The americium content in daily urinary and fecal output from male (FIG.17) and female (FIG. 18) rats indicate that the efficacy of the C2E2dose was not altered by fasting rats prior to their C2E2 treatment.

Analysis of the total decorporation achieved in the seven days aftercontamination for all the rats treated with C2E2 showed significanteffects for gender (F_((1,44))=17.54, p<0.001) and C2E2 dose(F_((2,44))=40.71, p<0.001) but not for Fed state at treatment(F_((1,44))=1.51, p=0.23), confirming the observations in FIGS. 17 and18. Interactions between effects were not considered significant andtherefore, for subsequent analysis ad libitum and fasted groups werecombined. Statistical analysis has not been applied to daily data.

Effect of Gender on ²⁴1 Am Decorporation:

Americium decorporation in male rats is significantly greater over theseven days of the study than in female rats (F_((1,60))=21.37, p<0.001;FIG. 19). The observed difference between genders is consistent acrossall the different treatment groups (untreated, three C2E2 doses andCa-DTPA) and is not statistically significant at any individualtreatment level.

Effect of C2E2 Dose on ²⁴¹Am Decorporation: C2E2 enhanced theelimination of americium in a dose dependent manner in male and femalerats. In male rats total decorporation was significantly increasedcompared to untreated controls at doses of 600 mg/kg and above (p<0.001)and increasing the dose from 600 mg/kg to 1000 mg/kg also significantlyincreased decorporation (p<0.05). A similar trend was observed in femalerats with doses above 600 mg/kg inducing significantly enhanceddecorporation (p<0.001), the 1000 mg/kg dose appears to be moreeffective than the 600 mg/kg dose although this did not reachstatistical significance.

Data Analysis of ²⁴¹Am Tissue Burden:

Tissues and organs were collected from all 64 rats that completed thestudy, of these 61 rats (32 male and 29 female) were included in dataanalysis. Three fasted female rats (1×600 mg/kg; 2×1000 mg/kg) wereexcluded from the dataset as they did not receive a complete dose.

Effect of Food on ²⁴¹Am Tissue Burden:

Americium burden in liver, wound site tissue were determined and theskeletal burden was estimated seven days after contamination in animalstreated with 200, 600, or 1000 mg/kg C2E2 one day after intramuscularcontamination. For all of the tissues no difference in americium burdenwas observed between rats with ad libitum access to food and rats fastedovernight prior to C2E2 treatment (Liver, F_((1,44))=0.02, p=0.88;Wound, F_((1,44))=0.84, p=0.37; Skeleton, F_((1,44))=0.02, p=0.88). Incontrast, in these animals gender was a significant factor with malerats having lower retention than female rats (Liver, F_((1,44))=72.97,p<0.001; Wound, F_((1,44))=12.87, p<0.01; Skeleton, F_((1,44))=48.29,p<0.001). C2E2 dose also influenced tissue burden (Liver,F_((2,44))=8.52, p<0.01; Wound, F_((2,44))=10.03, p<0.001; Skeleton,F_((1,44))=4.79, p<0.05). Interactions between effects were notconsidered significant and for subsequent analysis ad libitum and fastedgroups were combined.

Effect of Gender on ²⁴¹ Am Tissue Burden:

For all animals in the study, lower liver and wound site americiumburdens were observed in male rats than in female rats (Liver,F_((1,60))=91.24, p<0.001 and Wound, F_((1,60))=18.59, p<0.001).Although the trend for higher average americium retention at the woundsite in female rats was observed across all groups (FIG. 20) it onlyreached statistical significance in the 200 mg/kg C2E2 dose (p<0.05).Ca-DTPA and C2E2 reduced americium burden in male rat livers compared tofemale rat livers (14 mg/kg Ca-DTPA, p<0.05; 200 and 600 mg/kg C2E2,p<0.001; and 1000 mg/kg C2E2, p<0.05), americium burdens in untreatedrats were not significantly different between genders (p=0.42). Incontrast to the liver and wound site, the estimated skeletal burden waslower in the female rats than in male rats (F_((1,60))=58.51, p<0.001).In the Ca-DTPA groups the estimated skeletal burden was notsignificantly influenced by gender (p=1.00) for all other groupsestimates of female americium skeletal burden were lower than incomparable males (Untreated and 200 mg/kg C2E2, p<0.001; 600 and 1000mg/kg C2E2, p<0.05).

Effect of C2E2 Dose on ²⁴¹Am Tissue Burden:

In both male and female rats, C2E2 reduced the americium burden inliver, skeleton, and at the wound site. In male rats, the liver burdenwas significantly reduced compared to untreated controls at doses of 200mg/kg (p<0.05) and 1000 mg/kg (p<0.001). Although the liver burdenfollowing a 600 mg/kg C2E2 dose did not reach statistical significance(p=0.056) it is consistent with the trend of C2E2 treatment reducingliver burden. A dose-dependent reduction in ²⁴¹Am liver burden wasobserved in female rats. Only the reduction in burden following the 1000mg/kg dose was statistically significant compared to untreated controlsat the 1000 mg/kg dose (p<0.01). Estimated skeletal americium burden inmale rats decreased in a C2E2 dose dependent manner with significantreduction compared to untreated control animals at doses of 600 mg/kgand above. Skeletal burden in female rats was lower than in male ratsfor all groups and was not significantly changed by C2E2 treatment (FIG.21). Although overall C2E2 dose was a significant effect for wound siteretention of americium (F_((3,52))=7.92, p<0.01) this effect did notcorrespond with a significant change in tissue burden compared tountreated controls at any single C2E2 dose (FIG. 22). In female rats, atrend for reduced burden with increasing C2E2 dose may exist; C2E2appeared to have no effect on skeletal burden in male rats.

In conclusion, a single oral dose of C2E2 can induce significantimprovement in outcome based on primary measures—decorporation, liverburden and skeletal burden. C2E2 efficacy is independent of foodalthough it is dependent on gender; this dependence is consistent withthe differences between genders seen in untreated and Ca-DTPA treatedanimals and is due to a gender difference in the biokinetics ofamericium rather than a direct C2E2 effect.

Efficacy Studies in Dogs:

Dogs were administered ²⁴¹Am (target of 3 μCi/animal) by nose-onlyinhalation on Day 0. At 1 day post exposure, animals received one ofthree different oral doses (10, 300, 500 mg/kg) of C2E2. Group 1 was acontrol and was used for determining the unperturbed biokinetics.Fourteen (14) days post C2E2 administration, the animals were sedatedand euthanized. Preliminary urinary excretion and liver retention datawere obtained (Table 8). These results demonstrated that C2E2administered orally to contaminated dogs was able to enhance urinaryexcretion of the radionuclide and reduce liver burden.

TABLE 8 ²⁴¹Am recovery in urine and livers of dogs treated with C2E2orally. Total Recovered Activity (nCi) C2E2 Treat- Treat- Dose Genderment Gender ment Gender (mg/kg) Urine Mean Mean Liver Mean Mean M 014.14 13.47 15.17 326.2 230.03 189.93 M 0 12.80 133.9 F 0 13.95 16.86108.4 149.84 F 0 19.77 191.3 M 100 187.57 169.10 168.70 213.3 179.89168.91 M 100 150.63 146.5 F 100 193.85 168.30 186.4 157.92 F 100 142.75129.4 M 300 97.24 157.29 169.50 109.2 120.06 132.28 M 300 217.34 130.9 F300 121.32 181.71 87.6 144.49 F 300 242.10 201.4 M 500 288.78 274.65188.82 49.4 49.73 45.01 M 500 260.53 50.1 F 500 65.39 103.00 34.3 40.30F 500 140.61 46.3Toxicology10-Day Repeat-Dose Study in Dogs:

An exploratory study was conducted to evaluate the toxicity and todetermine the toxicokinetics of the test article, C2E2, whenadministered to dogs via 10 daily oral gavage doses. Groups of 2 Beagledogs/sex were administered C2E2 via oral gavage at dose levels of 0, 60,200, 400, or 600 mg/kg. The vehicle was reverse osmosis water and thedose volume was 8 mL/kg. Assessment of toxicity was based on mortality,clinical observations, body weight, food consumption, clinicalpathology, gross pathology, organ weights, histopathology of selectedtissues, and plasma essential trace elements analysis. Blood sampleswere collected on Days 1 and 10 for toxicokinetic evaluations of C2E2,C2E1, DTPA and C2E3. C2E3 is believed to be an impurity in the C2E2 lotused.

Two animals were sacrificed early due to moribund condition, one malegiven 600 mg/kg/day and one male given 400 mg/kg/day. These animals hadnotably higher plasma C2E2 concentrations than other animals in theirrespective dose groups. Based on clinical pathology findings andmicroscopic evidence of hepatocellular degeneration/necrosis, themoribund condition of these animals was due primarily to testarticle-related liver toxicity.

Animals given ≧200 mg/kg/day demonstrated two types of test articlerelated clinical observations. These observations consisted ofvomitus/emesis, typically immediately after dose administration, andfecal abnormalities (non-formed, liquid, or mucoid), which typicallyoccurred within 1 hour of dose administration. Clinical observationsresolved by the following morning. Dose-dependent body weight lossoccurred in males given ≧200 mg/kg/day and females given ≧400 mg/kg/day.No test article-related depletion of essential trace elements in plasma(Cr, Co, Cu, Fe, Mg, Mn, Mo, Se, and Zn) was observed in dogs followingoral administration of C2E2 at repeated doses up to 600 mg/kg/day for 10days. Clinical chemistry changes consistent with hepatotoxicity wereobserved at doses ≧400 mg/kg/day. In the animals that were sacrificedearly, these changes included markedly increased aspartateaminotransferase (AST) and alanine aminotransferase (ALT) activity inthe male given 600 mg/kg/day and sacrificed on Day 2, and markedlyincreased ALT activity in the male given 400 mg/kg/day and sacrificed onDay 6. In animals that survived to the scheduled necropsy on Day 11,mildly to moderately increased AST activity was observed on Day 6 inmales and females given 600 mg/kg/day, and on Day 11 in males given ≧400mg/kg/day and females given 600 mg/kg/day. Mildly to markedly increasedALT activity was observed on Days 6 and 11 of the dosing phase inanimals given ≧400 mg/kg/day.

Test article-related decreased absolute and relative liver/gall bladderweights occurred in females given 600 mg/kg/day and likely correlatedwith minimal hepatocyte degeneration/necrosis. Both males sacrificedearly exhibited slight (400 mg/kg/day) or severe (600 mg/kg/day)hepatocyte degeneration/necrosis (correlated to red to tan discolorationof the liver in the animal given 600 mg/kg/day). In addition, the liverof the animal given 400 mg/kg/day exhibited slight centrilobularcongestion/hemorrhage, minimal brown granular pigment, and minimal mixedinflammation. Both animals exhibited minimal (400 mg/kg/day) or slight(600 mg/kg/day) renal tubule cell degeneration/necrosis. The kidney ofthe animal given 400 mg/kg/day exhibited minimal basophilic tubules.Additional microscopic findings in these animals were noted in variousparts of the intestinal tract, stomach, lymph nodes, prostate, thymus,spleen, and gut-associated lymphoid tissue.

At the terminal sacrifice, test article-related microscopic findingswere present in the liver, kidney, and various segments of theintestinal tract. Hepatocyte degeneration/necrosis and mixed cellinflammation occurred in males given ≧200 mg/kg/day. In females,hepatocyte degeneration/necrosis was present at 600 mg/kg/day and mixedcell inflammation was present at ≧400 mg/kg/day. Both females given 600mg/kg/day had centrilobular congestion/hemorrhage, and several males andfemales had accumulations of pigment similar to that described in theunscheduled deaths. The male given 600 mg/kg/day had a single thrombosedblood vessel within the hepatic parenchyma. Based on minimal severityand lack of correlative differences in clinical pathology parameters,the liver findings in the male given 200 mg/kg/day were not consideredadverse; all other microscopic liver findings in males given 400 or 600mg/kg/day and females given 600 mg/kg/day were considered adverse.

In the kidney, tubule cell degeneration/necrosis was present in onefemale given 600 mg/kg/day and one female given 400 mg/kg/day; however,this finding was not present in males at the terminal sacrifice, but wasnoted in both males sacrificed early. Due to the severity of thefindings, these were considered adverse. Test article-related findingsin various segments of the intestinal tract included crypt/glandabscess, infiltrates of neutrophils, congestion, and/ordepletion/necrosis of lymphocytes of the GALT in one or more animalsgiven ≧200 mg/kg/day. Since these findings were sporadic, of minimalseverity, and possibly related to stress, they were not consideredadverse.

Exposure to C2E2 increased with increased C2E2 dose level from 60 to 600mg/kg/day. The increases in mean C_(max) and AUC₀₋₂₄ values were notconsistently dose proportional. Sex differences were generally less than2-fold in C2E2 mean C_(max) and AUC₀₋₂₄ values. No accumulation of C2E2was observed after multiple dosing of C2E2 in dogs. Ratios of C2E1,DTPA, and C2E3 AUC₀₋₂₄ values compared to that of C2E2 indicated thatsystemic exposure of C2E1, DTPA, and C2E3 were very low relative to C2E2(<1%). Daily administration of C2E2 to purebred beagle dogs by oralgavage for 10 days at dose levels of 60, 200, 400, and 600 mg/kg/dayresulted in the early sacrifice of one male given 600 mg/kg/day and onemale given 400 mg/kg/day due to body weight loss and declining health.Due to early sacrifices, the dose levels of 400 and 600 mg/kg/dayexceeded the maximum-tolerated dose. The liver and kidney wereidentified as target organs of toxicity at ≧400 mg/kg/day. Based onthese findings, the no observed adverse effect level (NOAEL) is 200mg/kg/day. After 10 days of dosing, administration of 200 mg/kg/day C2E2corresponded to mean C2E2 C_(max) values of 59,450 and 71,950 ng/mL andC2E2 AUC₀₋₂₄ values of 150,727 and 183,760 ng·hr/mL for males andfemales, respectively.

Example 2

The efficacy of C2E2 of Lot 050 obtained using Method 2 was evaluated inmale and female beagle dogs administered a nitrate complex ofamericium-241 (²⁴¹Am) via inhalation (INH). C2E2 was administered 24hours post ²⁴¹Am inhalation exposure. Animals were monitored and urineand feces were collected daily. Cages were rinsed daily. Animals wereeuthanized 14 days later and tissues collected. Tissues were processedand analyzed for ²⁴¹Am content. The in-life measurements and biokineticsof ²⁴¹Am with and without chelation treatment are described.

Sixteen (16) male and female beagle dogs underwent a 14-day quarantineperiod for acclimation. Once released from quarantine, animals wereweighed and that weight was used to randomize the animals into thestudy. Dogs were 13.3±2.2 months of age at study initiation and weighed8.9±1.2 kg. After randomization, the animals were acclimated tometabolism cages for approximately 24 hours prior to radionuclideinhalation administration of ²⁴¹Am(III)-nitrate at time 0 followed byoral gavage administration of vehicle (water) or 100, 300, or 500 mg/kgC2E2 at 24 hours post inhalation exposure. Animals were placed inmetabolism cages for the duration of the in-life phase.

Animals were euthanized 14 days after administration of ²⁴¹Am. A fullnecropsy was conducted and liver, spleen, kidneys, lungs and trachea,muscle samples (right and left quadriceps), GI tract (stomach andesophagus, upper and lower intestine), gonads, two femurs, lumbarvertebrae (L1-L4), paws and tail, TBLN, and all soft tissue remains werecollected. The brain and eyes were removed from the skull and combinedwith the soft tissue remains. The skeleton was defleshed and all bonesamples collected for analysis. Pelt was not analyzed for ²⁴¹Am content.

Urine, feces, cage rinse, and all tissue samples were processed by heatand chemical treatments. The samples were analyzed by gamma pulse heightanalysis.

Therapeutic C2E2 was successfully evaluated for decorporation efficacyin beagle dogs exposed by inhalation to ²⁴¹Am as a relatively solublenitrate. Urinary and fecal elimination profiles as well as tissueburdens compared to controls confirmed that the three therapeutic doselevels decorporated ²⁴¹Am when administered 24 hours after radionuclideadministration. The content of ²⁴¹Am in soft tissues and bone was alsoreduced significantly as a result of the decorporation therapy.

For the study, stock C2E2 (L/N: 020WJL050) was obtained as a whitepowder and stored at 2-8° C. The C2E2 was weighed out and dissolved invehicle, DI water. Solutions were prepared at 20 mg/mL, 60 mg/mL, and100 mg/mL on each day of dosing. The formulation was protected fromlight by wrapping the container in aluminum foil. Metal-free spatulas,glassware, and stir bars were used when weighing and preparing theformulation.

Americium-241 was obtained from the Department of Energy; it wasprocessed and formulated as a stock solution as a nitrate complex. Analiquot of 20 mCi of the nitrate complex was taken to dryness on amedium temperature hotplate. One hundred milliliters of 0.25M nitricacid was added. The pH was determined to be 0.74. An aliquot wascollected and analyzed by gamma pulse height analysis. The finalconcentration of the formulation was 201.1 μCi/mL.

Eight (8) male and eight (8) female beagle dogs were ordered fromCovance Laboratories for study assignment. Sixteen (16) animals wereassigned to one of four study groups by body weight stratification andrandomization. Dogs were 13.3±2.2 months of age at study initiation andweighed 8.8±1.2 kg. Study animals were conditioned to their metabolismcages for 24 hours prior to ²⁴¹Am administration. Observations wereconducted to ensure that animals were aware of food and water locationsas well as were excreting normal amounts of urine and feces. Animalswere uniquely identified with study groups by ear tattoo. In addition,each cage contained color-coded cage cards with the study-specific ID onit.

On the morning of exposure, animals were fasted and sedated withacepromazine (0.05 mg/kg) by intramuscular administration. Dogs wereanesthetized with isoflurane (5%). A latex mask was placed over themuzzle to minimize external contamination of the dog. The latex-coveredmuzzle was placed into the exposure plenum where a mixture of oxygen andisoflurane were continuously flowing and being monitored. Oxygen in thebox was maintained at 45-55% and isoflurane was delivered as needed(2-3% in oxygen) to ensure the dog remained anesthetized. Aerosols weregenerated from the ²⁴¹Am nitrate solution using a Hospitec nebulizeroperated at 10 psi. The aerosol was transited through a tube furnaceoperating at 70-80° C. to dry the aerosol and minimize the amount ofacid present.

The exhaust flow was 12.2 L/min. Aerosols were collected on Pallflex®FiberFilm™ filters at a flow rate of 0.49 L/min for filter samples and1.95 L/min for cascade impactor samples. Filters were placed in 20-mLvials with 5 mL of 7N HNO₃ and vortexed for 30 seconds. The filters wereremoved and placed singly into another 20-mL vial. A 50-4 aliquot wasremoved from the original vial. The filter and the aliquot wereindividually analyzed for ²⁴¹Am and the amount collected on the filterwas determined. The aerosol concentration was determined to be 1155±223nCi/L. The particle size was determined to be 0.63 μm AMAD (activitymedian aerodynamic diameter) with a 1.72 geometric standard deviation(GSD). Animals were exposed for 8 minutes. At the conclusion of eachexposure, the animals were removed from the exposure box, placed in atransport box and returned to their metabolism cages. The targetdeposited activity in the respiratory tract was 3 μCi.

Twenty-four (24) hours post radionuclide administration, animals wereadministered test article (or vehicle) by oral gavage as outlined inTable 9. Animals received a single dose of test article (or vehicle) oneday following americium exposure. The animal was manually restrained andan appropriately sized feeding tube was inserted into the esophagus. Thetherapeutic was delivered through the tube (33-52 mL) to achieve thedesired mass-normalized dosage of C2E2. Control dogs were administered45 mL of DI water. One (1) mL of water was flushed through the feedingtube to ensure complete delivery of test article. The animal wasreturned to its metabolism cage after every dose administration. A studyschedule is shown in Table 9.

TABLE 9 Experimental design ²⁴¹Am, test articles and necropsy. TestArticle Exposure Test Dose Dose Necropsy Group N Gender Article (mg/kg)Regimen Day 1001-1002, 2 M Water n/a Day 1 Day 14 1003-1004 2 F2001-2002, 2 M C2E2 100 Day 1 Day 14 2003-2004 2 F 3001-3002, 2 M C2E2300 Day 1 Day 14 3003-3004 2 F 4001-4002, 2 M C2E2 500 Day 1 Day 144003-4006 2 F

Upon confirmation of euthanasia, each dog underwent a full necropsy.Tissues collected included: liver, spleen, kidneys, lungs and trachea,muscle sample (right and left quadriceps), GI tract plus contents(stomach and esophagus, upper and lower intestine), gonads, two femurs,lumbar vertebrae (L1-L4), paws and tail, tracheobronchial lymph nodes(TBLN), and all soft tissue remains. The brain and eyes were removedfrom the skull and combined with the soft tissue remains. The skeletonwas defleshed and all bone samples collected for analysis. All tissuesamples were placed into appropriately sized and labeled specimencontainers with the exception of the pelt, which was not analyzed for²⁴¹Am.

All biological and cage-rinse samples (except pelt) were analyzed bygamma pulse height analysis. Prior to tissue-dependent analysis, sampleswere thermally and chemically processed by sample-specific methods.After samples were prepared they were placed into 20-mL liquidscintillation vials and counted on the gamma counter (Perkin Elmer, 2480Wizard2 Gamma Counter).

Mean and standard deviation of the body weights collected forrandomization and exposure data were performed using Microsoft Excel.Statistical analysis was conducted by one-way analysis of variance(ANOVA) used to evaluate the pattern of recovered doses. For each sampletype, differences between untreated controls and treated groups wereassessed with individual F-tests based on the ANOVA's pooled estimate ofunderlying between-animal variance. Absence of significant statisticalevidence of a difference in the pattern response across genders (p>0.05)accompanied by significant (p<0.05) evidence of overall differencesbetween genders provides evidence of constant shift in response betweengenders, irrespective of treatment.

The in-life portion of the study was accomplished without incident. Themajority of animals had no unusual or adverse effects related to thestudy; this includes vomiting from the C2E2 dose administrations.However, it was noted that one animal from the high level C2E2 dosegroup (Animal 4004) had foamy emesis in the cage pan after gavagedosing. Thus the fidelity of dose retention in this animal cannot beassured.

Table 10 summarizes the total activity of ²⁴¹Am recovered for the fourexperimental groups in this study. Group 4 recovered less material thanthe remaining groups but this was largely due to a single outlier (4003)that received less than a total of 1000 nCi. The reason for the lowactivity in this animal is unknown. All records show that there was noissue with processing of samples, no vomiting on study, the aerosolconcentration was on target, and the animal did not wake duringexposures. The only notable finding is that this dog weighed only 6.5 kgand was the smallest animal on study but this alone does not account forthe low activity received. If this animal is removed from the groupaverage, the group average increases to 1940 which is similar to theaverage from group 1 animals. For this study, animals received anaverage of 2220 nCi±610 total deposited activity. Unlike wound or IVstudies, fractional recoveries cannot be calculated with inhalationexposures because the actual delivered activity of ²⁴¹Am to therespiratory tract can only be reconstructed based on aerosolconcentration data together with physiological measurements orradiochemical measurement data. The estimated deposited activity isgiven by the equation:Deposited Activity=Aerosol Concentration (AC)*Respiratory Minute Volume(RMV)*Deposition Fraction (DF)*Exposure Duration (ED)

-   -   Where: AC=dog's aerosol conc (nCi/L); RMV=0.499*Body        Weight^(0.809); DF=10%; ED=8 min

The estimated delivered activity for the study was 2770 nCi for femalesand 2580 nCi for males. The delivered activity was approximately 11percent lower than the desired activity, which is well within theuncertainties of the exposure procedure. The deposition fraction for theparenchymal region of the lung is variable based on aerosolcharacteristics (particle size and σ_(g)) and breathing patterns and mayvary from the assumed 10%.

TABLE 10 Group recovered doses. Exposure Group Recovered Activity (nCi ±SD) 1001-1004 2020 ± 620 2001-2004 2430 ± 280 3001-3004 2720 ± 5604001-4004 1700 ± 550

FIGS. 23A and 23B show the daily urinary and fecal elimination of ²⁴¹Amfor all study groups. Statistical analysis has not been conducted on thedaily collections. Results are expressed as fraction of the totalrecovered ²⁴¹Am activity. All C2E2 dose groups displayed adose-dependent increase in urinary excretion compared to the untreatedcontrols. The urinary excretion of the dose groups was maintainedthrough the duration of the study. Fecal elimination was modestlyincreased for all dose groups through the first 3 days post exposurewhen the levels of elimination returned to control levels.

FIGS. 24A and 24B and Table 11 show the cumulative urine and cumulativefeces eliminations for all experimental groups. Treatment with all dosesof C2E2 resulted in a statistically significant increase of cumulativeactivity in urinary excretion compared to the untreated control group.The 500 mg/kg dose resulted in a higher urinary elimination of ²⁴¹Amcompared to the 100 and 300 mg/kg dose groups. Treatment with the midand high doses of C2E2 resulted in a statistically significant increaseof cumulative activity in fecal excretions compared to the untreatedcontrol group. Cumulative fecal excretion appeared to increase withincreasing doses of C2E2, but the response was not monotonic, i.e., thelargest amount of 241Am excreted occurred in the mid-dose group.Nevertheless, the amount of ²⁴¹Am excreted in feces was statisticallygreater than for the control group for the mid- and high-dose treatedgroups, and elevated (but not statistically significantly different) forthe low-dose group.

TABLE 11 Average percent recovered activity. Urine Feces Water  2.71(0.29) 26.53 (1.89) 100 mg/kg C2E2 22.35 (0.56)** 31.78 (3.21) 300 mg/kgC2E2 21.93 (2.26)** 41.85 (4.43)** 500 mg/kg C2E2 35.63 (0.70)** 36.53(1.18)* Mean (SEM); *p < 0.05 against Control group, **p < 0.01 againstControl group

The terminal contents of ²⁴¹Am in the various tissues analyzed are showngraphically for all experimental groups in FIGS. 25A-D and 26A-F, andnumerically in Tables 12 and 13. All results are expressed as percentageof the total recovered ²⁴¹Am activity. FIGS. 25A-D show tissue contentsfor liver, spleen, kidney, and lung for each dosing group. All threeC2E2 groups had statistically significant reductions in liver, kidneyand lung burdens. Little dose response was noted in the remaining spleenand kidney burden between the mid and high dose groups.

TABLE 12 Average percent recovered activity. Group Liver Spleen KidneyLung Water 27.20 (2.01) 0.30 (0.09) 0.77 (0.01) 13.98 (0.91)   100 mg/kg19.13 (1.34)** 0.16 (0.03) 0.37 (0.03)** 9.69 (1.77)*  C2E2 300 mg/kg13.35 (1.51)** 0.07 (0.02)** 0.28 (0.04)** 7.23 (0.89)** C2E2 500 mg/kg 7.71 (0.35)** 0.09 (0.02)* 0.29 (0.01)** 5.10 (0.31)** C2E2 Mean (SEM);*p < 0.05 against Control group, **p < 0.01 against Control group

TABLE 13 Average percent recovered activity. GIT Testes Ovaries TBLNSoft Tissue Total Bone Water 0.95 (0.09) 0.006 (0.001) 0.003 (0.001)0.035 (0.006) 2.05(0.12) 25.50 (2.16) 100 mg/kg C2E2 0.53 (0.05)** 0.003(0.001)* 0.001 (0.000) 0.028 (0.002) 1.08 (0.07)** 14.90 (1.97)** 300mg/kg C2E2 0.39 (0.06)** 0.003 (0.001)* 0.001 (0.001)* 0.024 (0.004)0.99 (0.12)** 13.93 (1.30)** 500 mg/kg C2E2 0.44 (0.05)** 0.003 (0.001)*0.001 (0.000) 0.023 (0.003) 1.01 (0.02)** 13.20 (0.86)** Mean (SEM): *p< 0.05 against Control group, **p < 0.01 against Control group

FIGS. 26A-F show terminal tissue contents for ovaries, testes, GIT,TBLN, soft tissue, and total bone for each dosing group compared withcontrols. In ovaries, 241Am contents for all dose groups were reducedcompared to the untreated control group. In testes, all treated groupsshowed statistically significant reductions in ²⁴¹Am content compared tountreated controls. There were no dose-dependent patterns indicated forthe ovary or testes. This may be attributed to the small amounts of²⁴¹Am present in the tissues. GIT (including contents) and soft tissue²⁴¹Am content both displayed statistically significant but notdose-dependent tissue reductions compared to controls. About 50% and 40%reductions of ²⁴¹Am content were observed for TBLN and total bonecontent, respectively. There was also a suggestion of a dose-dependentdecrease in terminal content of ²⁴¹Am, but the trend was not a strongone.

Table 14 shows the percentage tissue content reduction compared to theuntreated control group and Table 15 is the percentage of urinaryenhancement or fecal reduction compared to the untreated control group.The high-dose group showed >65% decrease in liver and spleen, >50%decrease in kidney, lung, GIT, gonad, and soft tissue burdens, and >30%decrease in TBLN and total bone content compared to untreated controls.The mid-dose treatment group showed a >60% decrease in spleen, kidney,and gonad burdens, >45% decrease in liver, lung, GIT, soft tissue andtotal bone, and >30% decrease in TBLN. The low treatment groupshowed >40% decreases in spleen, kidney, GIT, gonads, soft tissue, andtotal bone burdens and >20% decreases in liver, lungs, and TBLN burdens.

TABLE 14 Percent ⁴¹Am content reduction in tissue. Soft Total LiverSpleen Kidneys Lungs GIT Gonads TBLN Tissue Bone 100 mg/kg C2E2 29.747.7 52.4 30.6 43.9 51.1 22.9 47.1 41.6 300 mg/kg C2E2 51.0 74.8 63.048.2 58.2 66.3 31.7 51.8 45.5 500 mg/kg C2E2 71.7 69.0 61.8 63.5 54.155.3 35.0 50.7 48.3

TABLE 15 Percent change in excretion. Feces Urine 100 mg/kg C2E2 115 820300 mg/kg C2E2 155 805 500 mg/kg C2E2 135 1310

Urinary output increased over 800% for the low and mid dose groups and1300% for the high dose group compared to untreated controls. Fecaloutput increased over 110% for the low and high dose groups, and 150%for the mid dose group.

Urinary elimination profiles as well as tissue burdens compared tocontrols confirmed that the three therapeutic dose levels decorporated²⁴¹Am when administered 24 hours after radionuclide administration, andin many cases in a dose-dependent manner. The high-dose group of C2E2decorporated ²⁴¹Am more efficiently than the low-dose group. Future doseadministrations will focus on the higher levels of administered C2E2,although, while not wishing to be bound to any particular theory, it isspeculated that the modest increases in dose-dependent efficacy may becompensated effectively by multiple administrations of C2E2, much as isdone with DTPA therapy.

Example 3

The solubility of C2E2 of Lot 050 obtained using Method 2 was initiallydetermined to be 100 mg/mL. However, dosing solutions prepared at thisconcentration exhibited precipitation over time. The aim of this studywas to understand the process behind the initial super-saturation andsubsequent precipitation and to determine the solubility of C2E2.

A precipitation study was performed to estimate the concentration atwhich no precipitation occurs. Initially, 12 samples of C2E2 wereprepared at concentrations ranging from 40-150 mg/mL in pH 3.0 buffer insteps of 10 mg/mL. The solutions were left to stir until precipitationoccurred.

Three samples of C2E2 at 60, 70 and 100 mg/mL in pH 3.0 buffer wereprepared and stirred at room temperature. At 0.083, 5, 1, 2, 4, 6, 20,24 and 44 hours 0.5 mL samples were withdrawn centrifuged at 14,000×gand the concentration of supernatant measured by HPLC-CAD. At eachtime-point the pH of the supernatant was also measured.

After 24 hours, precipitation was observed in solutions with C2E2concentrations above 90 mg/mL. Precipitation was observed in solutionswith C2E2 at concentrations above 60 mg/mL that were left to stand atroom temperature with no stirring for 48 hours after 24 hours withstirring. The solubility of C2E2 at pH 3.0 is between 60 and 70 mg/mL.In the second part of the study, C2E2 at concentrations just above andbelow the determined solubility and a third sample at a higherconcentration were prepared to test whether C2E2 precipitation resultedin lower equilibrium solubility. The concentration time profile achievedis shown in FIG. 27. Solutions of C2E2 prepared at 60 and 70 mg/mLsolutions were stable after 6 hours and the 70 mg/ml sample was at ahigher concentration than the 60 mg/ml solution. Some precipitation ofthe 70 mg/ml was observed before the end of the study, so theconcentration shown by the line with the ▪ symbols for the 70 mg/mLsolution is the equilibrium solubility. For the concentration of the 100mg/ml solution, C2E2 remained in a supersaturated solution for 20 hoursand then suddenly precipitated out of solution between 20-24 hours, withthe final solubility in the range of the 60 and 70 mg/mL C2E2 solutions.The sudden change in concentration may indicate the formation of anotherpolymorph. The centrifuged solid was separated from the supernatant andanalysis by DSC will be performed.

Based on observation of dosing solutions, after a certain amount of timeprecipitation occurred in the C2E2 solutions accompanied by a lowermeasured solubility of C2E2.

Example 4

Metabolism of C2E2 by esterases in plasma may cause the loss of theester pro-moieties in C2E2. Ex vivo degradation of C2E2 in plasma, ifpresent, would have implications for the interpretation of C2E2 efficacydata and for the analysis of data from PK and TK studies; continuedmetabolism of the compound after blood samples have been collected wouldresult in the inaccurate calculation of PK parameters. Therefore, theaim of this study was to determine the stability of C2E2 in plasma.

Prior to the study, 975 μL of plasma (Rat, Beagle and Human) waspreheated to 37° C. in Eppendorff tubes and the test compound (C2E2) wasprepared in DI water (10 mg/mL). The C2E2 was from Lot 050 obtainedusing Method 2. To initiate the study, 25 μL of the C2E2 solution wasadded to the pre-heated plasma and briefly vortexed, to mix. At 0, 15,30, 60 and 120 minutes a 100 μL sample of plasma was taken, an equalvolume of cold acetonitrile was added and mixed to precipitate plasmaproteins. Samples were centrifuged at 4° C. and 14,000×g for 10 minutesto remove precipitated proteins, the supernatant was collected and theC2E2 concentration determined by HPLC-CAD. All conditions were repeatedin triplicate. Diltiazem was used as a positive control compound, andheat-inactivated plasma was used as a negative control.

HPLC Detection Methods: C2E2 analysis was performed on a prominence HPLC(Shimadzu Corporation, Kyoto, Japan) equipped with a Corona Ultracharged anion detector (CAD) (Thermo scientific, Sunnyvale, Calif.). Areverse phase gradient separation was used with an Alltima C18 column(250×2.1 mm² internal diameter with 5 μm particle size (Grace)) at 40°C. and a flow rate of 0.25 mL/min. The mobile phase is composed of waterwith 0.1% trifluoroacetic acid (A) and acetonitrile/isopropanol 2:1 (B).The mobile phase follows a linear gradient from 94:6 to 75:25 over 14min, the gradient then increases for 0.5 min to achieve a flow of 0:100for 3.5 minutes followed by re-equilibration of the system at 94:6 for 6min. The CAD analysis is performed at 25° C. with nitrogen flow at 35.1psi. For diltiazem, the gradient mobile phase consists of two majorcomponents: Mobile Phase A, aqueous TEA (0.2%, v/v) that was pH adjustedto 5.0 with o-PA, and Mobile Phase B, ACN.

The gradient is 0.01-34.99 min: 22% B; 35.00-44.99 min: 33% B;45.00-60.00 min: 38% B. The flow rate is 1.0 mL/min, a Thermo HypersilODS column (150 mm 4.6 mm i.d. with 5.0 μm particles) will be used,injection volume will be 10 μL and column oven temperature maintained at25° C. Diltiazem will be detected by UV at 240 nm. The limit ofquantification is 0.35 μg/mL. (Journal of Pharmaceutical Analysis 2012;2(3):226-237).

Preliminary results from the study are summarized in FIG. 28A-C.Although some variability was seen, a robust metabolism of the positivecontrol (diltiazem) was observed in all species. In all species tested,C2E2 metabolism was minimal or not detected for at least two hours;approximately 90% C2E2 remains after 2 hours in human plasma. As C2E2does not undergo metabolism, the observation that heat inactivation didnot prevent metabolism in the diltiazem positive control does not alterthe study conclusions.

Example 5

Preliminary PK/PD modeling predicted that C2E2 efficacy is correlatedwith AUC. While not wishing to be bound by any particular theory, if thetoxicology studies show that toxicity is associated with C_(max) ratherthan AUC then splitting the dose and administering it more frequentlycould increase the therapeutic window. To evaluate the benefits ofsingle vs. multiple daily doses of DTPA di-ethyl ester a decorporationstudy was performed in rats contaminated with ²⁴¹Am.

The animal study was conducted according to a protocol approved by TheUniversity of North Carolina at Chapel Hill Institutional Animal Careand Use Committee. Sixteen male Sprague Dawley rats were contaminatedwith ²⁴¹Am(NO₃)₃ (0.25 μCi) by intramuscular injection (0.1 mL, 0.1 MHNO₃) into the right hind leg, under isoflurane anesthesia. Immediatelyafter injection, body weights were recorded (Day 0 weight) and animalsplaced in individual metabolic cages. The 16 rats were assigned to fourgroups; untreated control, 5 daily oral gavage doses of 600 mg/kg C2E2once daily, 5 days of oral gavage treatment of 300 mg/kg C2E2 twicedaily or 5 daily intravenous doses of Zn-DTPA solution (13.3 mg/kg) viaa jugular vein catheter. The C2E2 was from Lot 050 obtained using Method2. The C2E2 dosing solution utilized was a 10% w/w solution in water.All animals were observed at least once daily for morbidity, mortality,and general appearance. Excreted urine and feces were collected at 2, 4,8, 10, 12, 14, 18, 20 and 24 hours after the first dose and daily forthe remainder of the study. The samples were transferred to 20 mLscintillation vials, weighed and placed in a gamma counter (Wizard22480, Perkin Elmer) for detection of ²⁴¹Am gamma activity. In addition,following necropsy, selected tissues were removed, weighed and placed ina gamma counter (Wizard2 2480, Perkin Elmer) for detection of ²⁴¹Amgamma activity. The total amount of ²⁴¹Am administered to the animalswas determined by quantifying the 59.7 keV photons emitted by ²⁴¹Am induplicate aliquots (100 μL) of the injection solution using a gammacounter (wizard series, Perkin-Elmer). A counting window from 40-80 keVand a 60-second counting time were used for acquisition and each readingwas corrected for background at acquisition. All experimental tissuesand samples were counted using the same gamma counter and protocol. Forall the samples, ²⁴¹Am content was expressed as a percentage of theinitial dose. The femur from the leg opposite to the injection site wasscaled by a factor of 20 to estimate total skeletal ²⁴¹Am burden.

All sixteen male rats completed the study. Preliminary analysis showsthat the elimination of americium was significantly enhanced in alltreatment groups compared to the untreated control and dividing the C2E2dosage and giving it twice daily did not significantly alter totaldecorporation. Table 16 shows the total decorporation in each group andFIG. 29 Error! Reference source not found. shows americium burdens inkey target tissues.

TABLE 16 Total decorporation in the seven days after americiumcontamination Total Decorporation Group (% of injected dose) UntreatedControl 17.7 ± 2.1 Daily C2E2 39.7 ± 2.6 Twice Daily C2E2 43.6 ± 1.6i.v. Control 54.0 ± 3.9

In addition to examining total decorporation and tissue burden, theprofile of americium decorporation in urine over time was obtained.FIGS. 30A and 30B show the profiles of urinary decorporation after onceand twice daily treatment with C2E2. An increase in decorporation afterthe second C2E2 dose can be clearly seen in FIG. 30B. The second peak inthe twice daily dosing group at around 14 hours results in greaterurinary elimination than the first at 2 hours, which may be due to someresidual C2E2 from the first dose. However, the time of day and theactivity of the animals may also affect the profiles as urine was notmanually expressed in this study. Over the course of the study, the oncedaily (OD) and twice daily (BD) groups resulted in very similar profilesof daily urinary elimination, consistent with the hypothesis that, whilenot wishing to be bound by any particular theory, drug AUC is the bestindicator of efficacy in the rat.

Preliminary analysis of the data suggests that splitting the C2E2 doseinto two smaller doses dose not decrease efficacy and therefore maypotentially increase the therapeutic window for C2E2. Full analysis willbe performed once the female arm of the study is complete.

Example 6

The objective of this study was to evaluate C2E2 for its ability toinduce reverse mutations at the histidine locus in Salmonellatyphimurium tester strains TA98, TA100, TA1535, and TA1537, and at thetryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in thepresence or absence of an exogenous mammalian metabolic activationsystem (S9).

C2E2 was evaluated in an initial mutagenicity assay in all five testerstrains at dose levels of 5.00, 16.0, 50.0, 160, 500, 1600, and 5000μg/plate with and without S9. C2E2 was from Lot 050 obtained usingMethod 2. C2E2 was prepared in Cell Culture Grade Water vehicle andformed a transparent colorless solution. Compared to concurrent vehiclecontrols, reductions in the mean numbers of revertant colonies was notedat the 5000 μg/plate level in TA100, TA1535, TA1537 and WP2uvrA underconditions without S9 and at ≧500 μg/plate in TA1535, TA1537 and WP2uvrAunder conditions with S9. Enhanced background lawn growth was observedat 5000 μg/plate with TA1537 in the test without S9. The reductions inthe numbers of revertant colonies and the enhanced background lawngrowth are indicative of C2E2 treatment-related toxicity. With exceptionto the enhanced background lawn in TA1537 without S9, all otherbackground lawns in all strains, with and without S9, were normalindicating there was appropriate bacterial growth during treatment toexpress a mutagenic event. There were no relevant increases in thenumbers of revertant colonies observed at any dose level with any strainin the absence or presence of S9 metabolic activation.

All vehicle and positive control values were within acceptable rangesand all criteria for a valid study were met.

These results indicate that C2E2 is negative in the Bacterial ReverseMutation Assay tested up to 5000 μg/plate with and without S9 and underthe conditions of this protocol.

Example 7

The objective of this in vitro assay was to evaluate the ability of C2E2to induce chromosomal aberrations in cultured Chinese hamster ovary(CHO) cells with and without an exogenous metabolic activation system.

C2E2 was prepared in cell culture grade water (CCGW) and formed acolorless, transparent, solution. The C2E2 was from Lot 050 obtainedusing Method 2. Subsequent stocks were prepared by serial dilution invehicle and all treatments were administered into 10 mL cultures in avolume of 10%. Vehicle control cultures were treated with 100 μL/mL perculture. The treatment periods were for 3 hours with and withoutmetabolic activation or approximately 20 hours without metabolicactivation.

In an initial chromosomal aberration assay (B1 test), C2E2concentrations of 6.92, 9.89, 14.1, 20.2, 28.8, 41.2, 58.8, 84.0, 120,172, 245, 350, and 500 μg/mL were tested in duplicate cultures in the 3-and 20-hour tests without S9 and in the 3-hour test with S9 metabolicactivation. All cultures, under all test conditions, were harvestedapproximately 20 hours from the initiation of treatment. At culturetermination, viable cells were counted and population doubling wascalculated for measurement of cytotoxicity to support selection of doselevels for aberration analysis. Visual observations of cultures forgeneral cell health and confluence were made prior to termination.

There was no relevant cytotoxicity observed in the 3-hour treatmenttests with and without S9 metabolic activation and the 245, 350 and 500μg/mL dose levels were selected for aberration analysis for each 3-hourtest condition. There were no statistically significant or biologicallyrelevant increases in the number of cells with chromosomal aberrationsobserved at any dose level examined in either 3-hour test underconditions with or without S9 metabolic activation.

In the 20-hour test without S9, a treatment-related decreasing trend incytotoxicty was shown by population doubling calculations. The 20.2, 172and 500 μg/mL treatment levels were selected for aberration analysisrepresenting 18% to 51% cytotoxicity measured by population doubling aspercent reductions of the vehicle control. Slides prepared from the 172and 500 μg/mL dose levels however, were absent of suitable metaphasecells for analysis. The reason for this was not determined and the testwas repeated to determine if the results were reproducible and if analternative method of measuring cytotoxicty was required.

The 20-hour test without S9 was repeated (B2 test) at the same doselevels as the initial B1 test. Results of the B2 test reproduced theinitial B1 test showing the same cytotoxicity profile measured bypopulation doubling, the same visual observations of numerous dividingcells at culture termination, and similar results of an absence ofmetaphase cells on prepared slides (245 and 500 μg/mL) at dose levelsestimated to be adequate for chromosomal analysis. Based on theseresults, mitotic indices were read from the B1 test slides to reevaluatecytotoxicity. Based on the mitotic indices, the 6.92, 9.89 and 14.1μg/mL dose levels were selected for aberration analysis. The 14.1 μg/mLdose level produced a 53% reduction in the mitotic index compared to theconcurrent vehicle control. Chromosomal analysis showed there were nobiologically relevant or statistically significant increases in thenumber of cells with aberrations observed at any dose level.

Under all test conditions, the vehicle control cultures were within thehistorical control range for cells with chromosomal aberrations and thepositive control cultures had significant increases in cells withchromosomal aberrations as compared with the vehicle control cultures.

C2E2 was determined to be negative for the induction of chromosomalaberrations under conditions with and without S9 when tested up tocytotoxicity limiting dose levels and the 500 μg/mL limit dose for thisassay.

Example 8

The objective of this study was to evaluate C2E2 for in vivo clastogenicactivity and/or disruption of the mitotic apparatus by detectingmicronuclei in polychromatic erythrocytes (PCE) in Sprague-Dawley ratbone marrow.

C2E2 was formulated in cell culture grade water vehicle and the dosevolume for all treatment groups was 20 mL/kg. The C2E2 was from Lot 050obtained using Method 2.

Male rats were administered vehicle control, or 500, 1000, or 2000mg/kg/day of C2E2 once a day for two days separated by approximately 24hours. The 2000 mg/kg high dose is the limit dose for this assayrecommended by ICH S2(R1) guidance. A positive control group of animalsreceived a single 60 mg/kg cyclophosphamide treatment on the second dayof dosing. Animals were observed at least twice daily for toxic signsand/or mortality. Bone marrow was extracted approximately 24 hours afterthe last treatment in all groups and at least 2000 PCEs per animal wereanalyzed for the frequency of micronuclei. Cytotoxicity was assessed byscoring the number of PCEs and normochromatic erythrocytes (NCEs)observed while scoring at least 500 erythrocytes per animal.

One 2000 mg/kg/day group animal was noted with audible respiration andhypoactive behavior on Day 2. There were no other adverse signs ofclinical toxicity observed in any other C2E2 treated animal. There wereno statistically significant decreases in C2E2 treated group PCE:NCEratios compared to the vehicle control value indicating an absence oftreatment-related bone marrow cytotoxicity. There were no statisticallysignificant or treatment-related increases in micronucleated PCEs at anyC2E2 dose level examined.

Under the conditions of this protocol, C2E2 was shown to be negative forinducing micronuclei in rat bone marrow when administered orally at 500,1000, or 2000 mg/kg/day once a day for 2 consecutive days.

Example 9

The protonation constants for C2E2 were determined by preparing a 5 mMsolution of free ligand in 0.15 M KCl. The constants were calculatedfrom three replicates, with each experiment consisting of a titrationwith acid, followed by a titration with base. After each addition oftitrant, a 30 second equilibration time passed before pH measurementwith a Seven Easy pH meter and (Ag/AgCl reference) glass electrode(Metler Tolledo). At the end of the titrations, the presence of C2E2 wasconfirmed by HPLC-CAD. Evaluation of the titration curve for C2E2 (FIG.31) identified 6 pK_(a) values that correspond with the three tertiaryamines and three carboxylic acids. The values were determined byrefinement using HYPERQUAD software. The calculated ionization constantsare shown in Table 17. The pKa values determined for C2E2 are consistentwith DTPA analogues previously investigated for use as MRI contrastagents with two carboxylic acids are functionalized such as DTPA-BMA andDTPA-BBA (Rizkalla, E. N., et al., Inorganic Chemistry, 32, 582-586,(1993) and Geraldes C. F., et al., Journal of the ChemicalSociety-Dalton Transactions, 327-335 (1995)).

TABLE 17 Acid dissociation constants for C2E2 and C2E1. Ligand pKa₆ pKa₅pKa₄ pKa₃ PKa₂ pKa₁ C2E2 1.45 ± 1.76 ± 1.87 ± 0.05 3.52 ± 0.03 4.68 ±0.02 9.40 ± 0.09 0.05 0.02 C2E1 <1.5 <1.5 1.18 ± 0.45 1.72 ± 1.5 2.99 ±0.05 6.35 ± 0.38

Example 10

The stability constant for a complex between C2E2 and gadolinium (i.e.,the C2E2-Gd complex) was determined by titration. C2E2 solutions (3-5mM) were prepared in 0.1 M KCl. Gadolinium chloride was added inequimolar concentrations. The experiment consisted of a titration withbase, followed by a titration with acid. The titration curves for freeC2E2 and C2E2-Gd are shown in FIG. 32. After each addition of titrant, a30 second equilibration time passed before pH measurement with a SevenEasy pH meter and (Ag/AgCl reference) glass electrode (Metler Tolledo).The pKa values previously determined, as described in Example 9, wereused to calculate the gadolinium stability constant using HYPERQUADsoftware. The stability of the C2E2-Gd complex was log K 17.34±1.13,which lies between those of EDTA and DTPA (17.0 and 22.5, respectively).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allpublications, patent applications, patents, patent publications, andother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

That which is claimed is:
 1. A polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid characterized by a powder x-ray diffraction pattern substantiallythe same as that shown in FIG. 6D and/or a powder x-ray diffractionpattern having peaks at about 8.1, 12.0, 13.8, 15.4, 16.0, 16.6, 18.3,19.3, 21.4, 22.1, 24.0, 26.5, and 29.2±0.2 degrees 2 theta, wherein thepolymorph has a melting point in a range from 133° C. to 141° C.
 2. Thepolymorph of claim 1, wherein the melting point is measured usingdifferential scanning calorimetry over a range of about 25° C. to about320° C. with a heating rate of about 10.00° C./min.
 3. A process ofpreparing the polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid of claim 1, comprising (a) combining DTPA bis-anhydride andabsolute ethanol to form a reaction mixture; (b) heating the reactionmixture to reflux while stirring for about 1.5 hours; (c) filtering thereaction mixture to form a filtrate; (d) cooling the filtrate to atemperature below about 20° C. to form a precipitate of a polymorph of6,9-bis(carboxymethyl)-3-(2-ethoxy-2-oxoethyl)-11-oxo-12-oxa-3,6,9-triazatetradecan-1-oicacid; (e) filtering the filtrate to obtain the precipitate; (f)optionally washing the precipitate with cold ethanol; (g) optionallywashing the precipitate with methyl tert-butyl ether to form a filtercake; (h) optionally mixing the filter cake with ethanol to form asecond slurry; (i) optionally heating the second slurry to a temperatureof about 70° C.; (j) optionally filtering the second slurry to form asecond filtrate; (k) optionally cooling the second filtrate to atemperature below about 20° C. to form a second precipitate; (l)optionally filtering the second filtrate to obtain the secondprecipitate; and (m) optionally drying the precipitate, therebyobtaining the polymorph.
 4. A method of treating a subject to remove aradioactive element from the subject comprising: administering atherapeutically effective amount of the polymorph of claim 1 to asubject, thereby removing the radioactive element from the subject. 5.The method of claim 4, wherein the administering step delivers to thesubject from about 1 mg to about 2,000 mg of the polymorph per kilogramof the subject's total body weight.
 6. The method of claim 4, whereinthe administering step is prior to the subject's exposure to aradioactive element.
 7. The method of claim 6, wherein the administeringstep is carried out to prevent incorporation of a radioactive elementinto the subject's tissues, organs, bones, or any combination thereof.8. The method of claim 4, wherein the administering step is after thesubject's exposure to a radioactive element.
 9. The method of claim 4,wherein the radioactive element comprises an isotope of plutonium (Pu),americium (Am), curium (Cm), or any combination thereof.
 10. The methodof claim 4, wherein the subject is a mammal.